Methods for the specific coagulation of vasculature

ABSTRACT

Disclosed are various compositions and methods for use in achieving specific blood coagulation. This is exemplified by the specific in vivo coagulation of tumor vasculature, causing tumor regression, through the site-specific delivery of a coagulant using a bispecific antibody.

This invention was made with government support under Contract No. P01HL 16411 by NIH. The government has certain rights in the invention.

The present application is a continuation-in-part of co-pending U.S.patent application Ser. No. 08/273,567, filed Jul. 11, 1994 nowabandoned; which is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 08/205,330, filed Mar. 2, 1994; which is acontinuation-in-part of U.S. Ser. No. 07/846,349, filed Mar. 5, 1992 nowabandoned.

BACKGROUND OF THE INVENTION

The entire text and figures of the above-referenced disclosures arespecifically incorporated herein by reference without disclaimer.

1. Field of the Invention

The present invention relates generally to the fields of blood vesselsand of coagulation. More particularly, it provides a variety of growthfactor-based and immunological reagents, including bispecificantibodies, for use in achieving specific coagulation.

2. Description of the Related Art

Advances in the chemotherapy of neoplastic disease have been realizedduring the last 30 years. This includes some progress in the developmentof new chemotherapeutic agents and, more particularly, the developmentof regimens for concurrent administration of drugs. A significantunderstanding of the neoplastic processes at the cellular and tissuelevel, and the mechanism of action of basic antineoplastic agents, hasalso allowed advances in the chemotherapy of a number of neoplasticdiseases, including choriocarcinoma, Wilm's tumor, acute leukemia,rhabdomyosarcoma, retinoblastoma, Hodgkin's disease and Burkitt'slymphoma. Despite the advances that have been made in a few tumors,though, many of the most prevalent forms of human cancer still resisteffective chemotherapeutic intervention.

A significant underlying problem that must be addressed in any treatmentregimen is the concept of "total cell kill." This concept holds that inorder to have an effective treatment regimen, whether it be a surgicalor chemotherapeutic approach or both, there must be a total cell kill ofall so-called "clonogenic" malignant cells, that is, cells that have theability to grow uncontrolled and replace any tumor mass that might beremoved. Due to the ultimate need to develop therapeutic agents andregimens that will achieve a total cell kill, certain types of tumorshave been more amenable than others to therapy. For example, the softtissue tumors (e.g., lymphomas), and tumors of the blood andblood-forming organs (e.g., leukemias) have generally been moreresponsive to chemotherapeutic therapy than have solid tumors such ascarcinomas.

One reason for the susceptibility of soft and blood-based tumors tochemotherapy is the greater physical accessibility of lymphoma andleukemic cells to chemotherapeutic intervention. Simply put, it is muchmore difficult for most chemotherapeutic agents to reach all of thecells of a solid tumor mass than it is the soft tumors and blood-basedtumors, and therefore much more difficult to achieve a total cell kill.Increasing the dose of chemotherapeutic agents most often results intoxic side effects, which generally limits the effectiveness ofconventional anti-tumor agents.

The strategy to develop successful antitumor agents involves the designof agents that will selectively kill tumor cells, while exertingrelatively little, if any, untoward effects against normal tissues. Thisgoal has been elusive to achieve, though, in that there are fewqualitative differences between neoplastic and normal tissues. Becauseof this, much research over the years has focused on identifyingtumor-specific "marker antigens" that can serve as immunological targetsboth for chemotherapy and diagnosis. Many tumor-specific, orquasi-tumor-specific ("tumor-associated"), markers have been identifiedas tumor cell antigens that can be recognized by specific antibodies.Unfortunately, it is generally the case that tumor specific antibodieswill not in and of themselves exert sufficient antitumor effects to makethem useful in cancer therapy.

More recently, immunotoxins have been employed in an attempt toselectively target cancer cells. Immunotoxins are conjugates of aspecific targeting agent, typically a tumor-directed antibody orfragment, with a cytotoxic agent, such as a toxin moiety. The targetingagent is designed to direct the toxin to cells carrying the targetedantigen and to kill such cells. "Second generation" immunotoxins havenow been developed, for example, those that employ deglycosylated ricinA chain to prevent entrapment of the immunotoxin by the liver and reducehepatotoxicity (Blakey et al., 1987a;b), and those with new crosslinkersto endow the immunotoxins with higher in vivo stability (Thorpe et al.,1988).

Immunotoxins have proven effective at treating lymphomas and leukemiasin mice (Thorpe et al., 1988; Ghetie et al., 1991; Griffin et al.,1988a;b) and in man (Vitetta et al., 1991). However, lymphoid neoplasiasare particularly amenable to immunotoxin therapy because the tumor cellsare relatively accessible to blood-borne immunotoxins. Also, it ispossible to target normal lymphoid antigens because the normallymphocytes, which are killed along with the malignant cells duringtherapy, are rapidly regenerated from progenitors lacking the targetantigens.

In contrast with their efficacy in lymphomas, immunotoxins have provedrelatively ineffective in the treatment of solid tumors (Weiner et al.,1989; Byers et al., 1989). The principal reason for this is that solidtumors are generally impermeable to antibody-sized molecules: specificuptake values of less than 0.001% of the injected dose/g of tumor arenot uncommon in human studies (Sands et al., 1988; Epenetos et al.,1986). Another significant problem is that antigen-deficient mutants canescape being killed by the immunotoxin and regrow (Thorpe et al., 1988).

Furthermore, antibodies that enter the tumor mass do not distributeevenly for several reasons. Firstly, the dense packing of tumor cellsand fibrous tumor stromas present a formidable physical barrier tomacromolecular transport and, combined with the absence of lymphaticdrainage, create an elevated interstitial pressure in the tumor corewhich reduces extravasation and fluid convection (Baxter et al., 1991;Jain, 1990). Secondly, the distribution of blood vessels in most tumorsis disorganized and heterogeneous, so some tumor cells are separatedfrom extravasating antibody by large diffusion distances (Jain, 1990).Thirdly, all of the antibody entering the tumor may become adsorbed inperivascular regions by the first tumor cells encountered, leaving noneto reach tumor cells at more distant sites (Baxter et al., 1991; Kennelet al., 1991).

Thus, it is quite clear that a significant need exists for thedevelopment of novel strategies for the treatment of solid tumors. Oneapproach involves the targeting of agents to the vasculature of thetumor, rather than to tumor cells. Solid tumor growth is highlydependent on the vascularization of the tumor and the growth of tumorcells can only be maintained if the supply of oxygen, nutrients andother growth factors and the efflux of metabolic products aresatisfactory. Indeed, it has been observed that many existing therapiesmay already have, as part of their action, a vascular-mediated mechanismof action (Denekamp, 1990).

The present inventors propose that targeting the vasculature will likelydeprive the tumor of life sustaining events and result in reduced tumorgrowth rate or tumor cell death. This approach is contemplated to offerseveral advantages over direct targeting of tumor cells. Firstly, thetarget cells are directly accessible to intravenously administeredtherapeutic agents, permitting rapid localization of a high percentageof the injected dose (Kennel et al., 1991). Secondly, since eachcapillary provides oxygen and nutrients for thousands of cells in itssurrounding `cord` of tumor, even limited damage to the tumorvasculature could produce an avalanche of tumor cell death (Denekamp,1990; Denekamp, 1984). Finally, the outgrowth of mutant endothelialcells, lacking a target antigen, is unlikely because they are normalcells.

At the present time, it is generally accepted that for tumor vasculartargeting to succeed, antibodies are required that recognize tumorendothelial cells but not those in normal tissues. Although severalantibodies have been raised (Duijvestijn et al., 1987; Hagemeier et al.,1986; Bruland et al., 1986; Murray et al., 1989; Schlingemann et al.,1985), none have shown a high degree of specificity. Also, there do notappear to be reports of any particular agents, other than theaforementioned toxins, that show promise as the second agent in avascular targeted antibody conjugate. Thus, unfortunately, whilevascular targeting presents certain theoretical advantages, effectivestrategies incorporating these advantages have yet to be developed.

SUMMARY OF THE INVENTION

The present invention overcomes the limitations of the prior art byproviding novel compositions and methods for use in achieving specificcoagulation, for example, coagulation in tumor vasculature, withlimiting side-effects. The invention, in a general and overall sense,concerns various novel immunological and growth factor-based bispecificcompositions capable of stimulating coagulation in disease-associatedvasculature, and methods for their preparation and use.

The invention provides binding ligands that may generally be describedas "bispecific binding ligands". Such ligands comprise a "first bindingregion" that typically binds to a disease-related target cell, such as atumor cell, or to a component associated with such a cell; to somecomponent associated with disease-related vasculature, e.g., tumorvasculature; or to a component of, or associated with,disease-associated stroma. The first binding region is operativelyassociated with or linked to a "coagulating agent", which may be eithera coagulation factor itself or may be a second binding region that iscapable of binding to a coagulation factor.

The binding ligands of the invention are described as "bispecific" asthey are "at least" bispecific, i.e., they comprise, at a minimum, twofunctionally distinct regions. Compositions and methods using otherconstructs, such as trispecific and mutlispecific binding ligands, arealso included within the scope of the invention. Combined compositions,kits and methods of using the bispecific coagulating ligands describedherein in conjunction with other effectors, such as other immunological-and growth-factor-based compositions, antigen-inducing agents,immunostimulants, immunosuppressants, chemotherapeutic drugs, and thelike, are also contemplated.

The first binding regions, and any second binding regions, may beantibodies or fragments thereof. As used herein, the term "antibody" isintended to refer broadly to any immunologic binding agent such as IgG,IgM, IgA, IgD and IgE. Generally, IgG or IgM are preferred because theyare the most common antibodies in the physiological situation andbecause they are most easily made in a laboratory setting. Monoclonalantibodies (MAbs) are recognized to have certain advantages, e.g.,reproducibility and large-scale production, and their use is generallypreferred. Engineered antibodies, such as recombinant antibodies andhumanized antibodies, also fall within the scope of the invention.

Where antigen binding regions of antibodies are employed as the bindingand targeting agent, a complete antibody molecule may be employed.Alternatively, a functional antigen binding region may be used, asexemplified by Fv, scFv (single chain Fv), Fab', Fab, Dab or F(ab')₂fragment of an antibody. The techniques for preparing and using variousantibody-based constructs are well known in the art and are furtherdescribed herein.

The coagulation factor portion of the binding ligands is formed so thatit maintains significant functional capacity, i.e., it is in a form sothat, when delivered to the target region, it still retains its abilityto promote blood coagulation or clotting. However, in certainembodiments, the coagulation factor portion of the binding ligands willbe less active than, for example, the natural counterpart of thecoagulant, and the factor will achieve the desired level of activityonly upon delivery to the target area. One such example is a vitaminK-dependent coagulation factor that lacks the Gla modification, whichwill nonetheless achieve significant functional activity upon binding ofthe first binding region of the bispecific ligand to a membraneenvironment.

Where a second binding region is used to bind a coagulation factor, itis generally chosen so that it recognizes a site on the coagulationfactor that does not significantly impair its ability to inducecoagulation. Likewise, where a coagulation factor is covalently linkedto a first binding agent, a site distinct from its functionalcoagulating site is generally used to join the molecules.

The "first binding region" of the bispecific ligands of the inventionmay be any component that binds to a designated target site, i.e., asite associated with a tumor region or other disease site in whichcoagulation is desired. The target molecule, in the case of tumortargeting, will generally be present at a higher concentration in thetumor site than in non-tumor sites. In certain preferred embodiments,the targeted molecules, whether associated with tumor cells, tumorvascular cells, tumor-associated stroma, or other components, will berestricted to such cells or other tumor-associated entities, however,this is not a requirement of the invention.

In this regard, it should be noted that tumor vasculature is`prothrombotic` and is predisposed towards coagulation. It is thuscontemplated that a targeted coagulant is likely to preferentiallycoagulate tumor vasculature while not coagulating normal tissuevasculature, even if other normal cells or body components,particularly, the normal endothelial cells or even stroma, expresssignificant levels of the target molecule. This approach is thereforeenvisioned to be safer for use in humans, e.g., as a means of treatingcancer, than that of targeting a toxin to tumor vasculature.

In certain embodiments, the first binding regions contemplated for usein this invention may be directed to a tumor cell component or to acomponent associated with a tumor cell. In targeting generally to atumor cell, it is believed that the first binding ligand will cause thecoagulation factor component of the bispecific binding ligand toconcentrate on those perivascular tumor cells nearest to the bloodvessel and thus trigger coagulation of tumor blood vessels, giving thebispecific binding ligand significant utility.

A first binding region may therefore be a component, such as an antibodyor other agent, that binds to a tumor cell. Agents that "bind to a tumorcell" are defined herein as ligands that bind to any accessiblecomponent or components of a tumor cell, or that bind to a componentthat is itself bound to, or otherwise associated with, a tumor cell, asfurther described herein.

The majority of such tumor-binding ligands are contemplated to beagents, particularly antibodies, that bind to a cell surface tumorantigen or marker. Many such antigens are known, as are a variety ofantibodies for use in antigen binding and tumor targeting. The inventionthus includes first binding regions, such as antigen binding regions ofantibodies, that bind to an identified tumor cell surface antigen, suchas those listed in Table I, and first binding regions thatpreferentially or specifically bind to an intact tumor cell, such asbinding to a tumor cell listed in Table II.

Currently preferred examples of tumor cell binding regions are thosethat comprise an antigen binding region of an antibody that binds to thecell surface tumor antigen p185^(HER2), milk mucin core protein, TAG-72,Lewis a or carcinoembryonic antigen (CEA). Another group of currentlypreferred tumor cell binding regions are those that comprise an antigenbinding region of an antibody that binds to a tumor-associated antigenthat binds to the antibody 9.2.27, OV-TL3, MOv18, B3, KS1/4, 260F9 orD612.

The antibody 9.2.27 binds to high M_(r) melanoma antigens, OV-TL3 andMOv18 both bind to ovarian-associated antigens, B3 and KS1/4 bind tocarcinoma antigens, 260F9 binds to breast carcinoma and D612 binds tocolorectal carcinoma. Antigen binding moieties that bind to the sameantigen as D612, B3 or KS1/4 are particularly preferred. D612 isdescribed in U.S. Pat. No. 5,183,756, and has ATCC Accession No. HB9796; B3 is described in U.S. Pat. No. 5,242,813, and has ATCC AccessionNo. HB 10573; and recombinant and chimeric KS1/4 antibodies aredescribed in U.S. Pat. No. 4,975,369; each incorporated herein byreference.

In tumor cell targeting, where the tumor marker is a component, such asa receptor, for which a biological ligand has been identified, theligand itself may also be employed as the targeting agent, rather thanan antibody. Active fragments or binding regions of such ligands mayalso be employed.

First binding regions for use in the invention may also be componentsthat bind to a ligand that is associated with a tumor cell marker. Forexample, where the tumor antigen in question is a cell-surface receptor,tumor cells in vivo will have the corresponding biological ligand, e.g.,hormone, cytokine or growth factor, bound to their surface and availableas a target. This includes both circulating ligands and "paracrine-type"ligands that may be generated by the tumor cell and then bound to thecell surface.

The present invention thus further includes first binding regions, suchas antibodies and fragments thereof, that bind to a ligand that binds toan identified tumor cell surface antigen, such as those listed in TableI, or that preferentially or specifically binds to one or more intacttumor cells. Additionally, the receptor itself, or preferably anengineered or otherwise soluble form of the receptor or receptor bindingdomain, could also be employed as the binding region of a bispecificcoagulating ligand.

In further embodiments, the first binding region may be a component thatbinds to a target molecule that is specifically or preferentiallyexpressed in a disease site other than a tumor site. Exemplary targetmolecules associated with other diseased cells include, for example, PSAassociated with Benign Prostatic Hyperplasia (BPH) and FGF associatedwith proliferative diabetic retinopathy. It is believed that an animalor patient having one of the above diseases would benefit from thespecific induction of coagulation in the disease site.

This is the meaning of "diseased cell" in the present context, i.e., itis a cell that is connected with a disease or disorder, which cellexpresses, or is otherwise associated with, a targetable component thatis present at a higher concentration in the disease sites and cells incomparison to its levels in non-diseased sites and cells. This includestargetable components that are associated with the vasculature in thedisease sites.

Exemplary first binding regions for use in targeting and delivering acoagulant to other disease sites include antibodies, such as anti-PSA(BPH), and GF82, GF67 and 3H3, that bind to FGF. Biological bindingligands, such as FGF, that bind to the relevant receptor, in this casethe FGF receptor, may also be used. Antibodies against vascular targetsmay also be employed, as described below. The targeting of the stroma orendothelial cells provides a powerful means of treating other diseaseswhere the "diseased cell" itself may not be associated with a strong orunique marker antigen.

In further embodiments, the first binding regions of the invention willbe components that are capable of binding to a component ofdisease-associated vasculature, i.e., a region of vasculature in whichspecific coagulation would be advantageous to the animal or patient.First binding regions capable of binding to a component specifically orpreferentially associated with tumor vasculature are currentlypreferred. "Components of tumor vasculature" include both tumorvasculature endothelial cell surface molecules and any components, suchas growth factors, that may be bound to these cell surface receptors ormolecules.

Certain preferred binding ligands are antibodies, and fragments thereof,that bind to cell surface receptors and antibodies that bind to thecorresponding biological ligands of these receptors. Exemplaryantibodies are those that bind to MHC Class II proteins, VEGF/VPFreceptors, FGF receptors, TGFβ receptors, a TIE (tyrosinekinase-immunoglobulin-epidermal growth factor-like receptor, includingTIE-1 and TIE-2), VCAM-1, P-selectin, E-selectin, α_(v) β₃ integrin,pleiotropin, endosialin and endoglin.

First binding regions that comprise an antigen binding region of anantibody that binds to endoglin are one group of preferred agents. Theseare exemplified by antibodies and fragments that bind to the sameepitope as the monoclonal antibody TEC-4 or the monoclonal antibodyTEC-11.

Antigen binding region of antibodies that bind to the VEGF receptor areanother group of preferred agents. These are particularly exemplified byantibodies and fragments that bind to the same epitope as the monoclonalantibody 3E11, 3E7, 5G6, 4D8, 10B10 or TEC-110. Anti-VEGF antibodieswith binding specificities substantially the same as any one of theantibodies termed 1B4, 4B7, 1B8, 2C9, 7D9, 12D2, 12D7, 12E10, 5E5, 8E5,5E11, 7E11, 3F5, 10F3, 1F4, 2F8, 2F9, 2F10, 1G6, 1G11, 3G9, 9G11, 10G9,GV97, GV39, GV97γ, GV39γ, GV59 or GV14 may also be used. Furthersuitable anti-VEGF antibodies include 4.6.1., A3.13.1, A4.3.1 and B2.6.2(Kim et. al., 1992); SBS94.1 (Oncogene Science); G143-264 and G143-856(Pharmingen).

Further useful antibodies are those that bind to a ligand that binds toa tumor vasculature cell surface receptor. Antibodies that bind toVEGF/VPF, FGF, TGFβ, a ligand that binds to a TIE, a tumor-associatedfibronectin isoform, scatter factor, hepatocyte growth factor (HGF),platelet factor 4 (PF4), PDGF (including PDGFa and PDGFb) and TIMP (atissue inhibitor of metalloproteinases, including TIMP-1, TIMP-2 andTIMP-3) are therefore useful in these embodiments, with antibodies thatbind to VEGF/VPF, FGF, TGFβ, a ligand that binds to a TIE or atumor-associated fibronectin isoform often being preferred.

In still further embodiments, it is contemplated that markers specificfor tumor vasculature may be those that have been first induced, i.e.,their expression specifically manipulated by the hand of man, allowingsubsequent targeting using a binding ligand, such as an antibody.

Exemplary inducible antigens include those inducible by a cytokine,e.g., IL-1, IL-4, TNF-α, TNF-β or IFN-γ, as may be released bymonocytes, macrophages, mast cells, helper T cells, CD8-positiveT-cells, NK cells or even tumor cells. Examples of the induced targetsare E-selectin, VCAM-1, ICAM-1, endoglin and MHC Class II antigens. Whenusing MHC Class II induction, the suppression of MHC Class II in normaltissues is generally required, as may be achieved using a cyclosporin,such as Cyclosporin A (CsA), or a functionally equivalent agent.

Further inducible antigens include those inducible by a coagulant, suchas by thrombin, Factor IX/IXa, Factor X/Xa, plasmin or ametalloproteinase (matrix metalloproteinase, MMP). Generally, antigensinducible by thrombin will be used. This group of antigens includesP-selectin, E-selectin, PDGF and ICAM-1, with the induction andtargeting of P-selectin and/or E-selectin being generally preferred.

Antibodies that bind to epitopes that are present on ligand-receptorcomplexes, but absent from both the individual ligand and receptor mayalso be used. Such antibodies will recognize and bind to aligand-receptor complex, as presented at the cell surface, but will notbind to the free ligand or uncomplexed receptor. A "ligand-receptorcomplex", as used herein, therefore refers to the resultant complexproduced when a ligand, such as a growth factor, specifically binds toits receptor, such as a growth factor receptor. This is exemplified bythe VEGF/VEGF receptor complex.

It is envisioned that such ligand-receptor complexes will likely bepresent in a significantly higher number on tumor-associated endothelialcells than on non-tumor associated endothelial cells, and may thus betargeted by anti-complex antibodies. Anti-complex antibodies includethose antibodies and fragments thereof that bind to the same epitope asthe monoclonal antibody 2E5, 3E5 or 4E5.

In further embodiments, the first binding regions contemplated for usein this invention will bind to a component of disease-associated stroma,such as a component of tumor-associated stroma. This includes antigenbinding regions of antibodies that bind to basement membrane components,activated platelets and inducible tumor stroma components, especiallythose inducible by a coagulant, such as thrombin. "Activated platelets"are herein defined as a component of tumor stroma, one reason for whichbeing that they bind to the stroma when activated.

Preferred targetable elements of tumor-associated stroma are currentlythe tumor-associated fibronectin isoforms. Fibronectin isoforms areligands that bind to the integrin family of receptors. Tumor-associatedfibronectin isoforms are available, e.g., as recognized by the MAb BC-1.This Mab, and others of similar specificity, are therefore preferredagents for use in the present invention. Fibronectin isoforms, althoughstromal components, bind to endothelial cells and may thus be consideredas a targetable vascular endothelial cell-bound ligand in the context ofthe invention.

Another group of preferred anti-stromal antibodies are those that bindto RIBS, the receptor-induced binding site, on fibrinogen. RIBS is atargetable antigen, the expression of which in stroma is dictated byactivated platelets. Antibodies that bind to LIBS, the ligand-inducedbinding site, on activated platelets are also useful.

One further group of useful antibodies are those that bind to tenascin,a large molecular weight extracellular glycoprotein expressed in thestroma of various benign and malignant tumors. Antibodies such as thosedescribed by Shrestha et. al. (1994) and 143DB7C8, described by Tuominen& Kallioinen (1994), may thus be used as the binding portions of thecoaguligands.

"Components of disease- and tumor-associated stroma" include variouscell types, matrix components, effectors and other molecules orcomponents that may be considered, by some, to be outside the narrowestdefinition of "stroma", but are nevertheless targetable entities thatare preferentially associated with a disease region, such as a tumor.

Accordingly, the first binding region may be an antibody or ligand thatbinds to a smooth muscle cell, a pericyte, a fibroblast, a macrophage,an infiltrating lymphocyte or leucocyte. First binding regions may alsobind to components of the connective tissue, and include antibodies andligands that bind to, e.g., fibrin, proteoglycans, glycoproteins,collagens, and anionic polysaccharides such as heparin and heparin-likecompounds.

In other preferred embodiments, the vasculature and stroma bindingligands of the invention will be binding regions that are themselvesbiological ligands, or portions thereof, rather than an antibodies."Biological ligands" in this sense will be those molecules that bind toor associate with cell surface molecules, such as receptors, that areaccessible in the stroma or on vascular cells; as exemplified bycytokines, hormones, growth factors, and the like. Any such growthfactor or ligand may be used so long as it binds to thedisease-associated stroma or vasculature, e.g., to a specific biologicalreceptor present on the surface of a tumor vasculature endothelial cell.

Suitable growth factors for use in these aspects of the inventioninclude, for example, VEGF/VPF (vascular endothelial growthfactor/vascular permeability factor), FGF (the fibroblast growth factorfamily of proteins), TGFβ (transforming growth factor B), a ligand thatbinds to a TIE, a tumor-associated fibronectin isoform, scatter factor,hepatocyte growth factor (HGF), platelet factor 4 (PF4), PDGF (plateletderived growth factor), TIMP or even IL-8, IL-6 or Factor XIIIa.VEGF/VPF and FGF will often be preferred.

Targeting an endothelial cell-bound component, e.g., a cytokine orgrowth factor, with a binding ligand construct based on a known receptoris also contemplated. Generally, where a receptor is used as a targetingcomponent, a truncated or soluble form of the receptor will be employed.In such embodiments, it is particularly preferred that the targetedendothelial cell-bound component be a dimeric ligand, such as VEGF. Thisis preferred as one component of the dimer will already be bound to thecell surface receptor in situ, leaving the other component of the dimeravailable for binding the soluble receptor portion of the bispecificcoagulating ligand.

The use of bispecific, or tri- or multi-specific, ligands that includeat least one targeting region capable of binding to a component ofdisease-associated vasculature has the advantage that vascularendothelial cells, and disease-associated agents such as activatedplatelets, are similar in different diseases, and particularly indifferent tumors. This phenomenon makes it feasible to treat numerousdiseases and types of cancer with one pharmaceutical, rather than havingto tailor the agent to each individual disease or specific tumor type.

The compositions and methods of the present invention are thus suitablefor use in treating both benign and malignant diseases that have avascular component. Such vasculature-associated diseases include benigngrowths, such as BPH, diabetic retinopathy, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, neovascularglaucoma and psoriasis. Also included within this group are synovitis,dermatitis, endometriosis, angiofibroma, rheumatoid arthritis,atherosclerotic plaques, corneal graft neovascularization, hemophilicjoints, hypertrophic scars, osler-weber syndrome, pyogenic granulomaretrolental fibroplasia, scleroderma, trachoma, and vascular adhesions.Each of the above diseases are known to have a common angio-dependentpathology, it is thus contemplated that achieving coagulation in thedisease site would prove beneficial.

The bispecific binding ligand-coagulation factor conjugates of thepresent invention may be conjugates in which the two or more componentsare covalently linked. For example, by using a biochemical crosslinkerand, preferably, one that has reasonable stability in blood, asexemplified by SMPT. The components may also be linked using thewell-known avidin (or streptavidin) and biotin combination. Variouscross-linkers, avidin:biotin compositions and combinations, andtechniques for preparing conjugates, are well known in the art and arefurther described herein.

Alternatively, such bispecific coagulating agents may be fusion proteinsprepared by molecular biological techniques, i.e., by joining a gene (orcDNA) encoding a binding ligand or region to a gene (or cDNA) encoding acoagulation factor. This is well known in the art and is furtherdescribed herein. Typically, an expression vector is prepared thatcomprises, in the same reading frame, a DNA segment encoding the firstbinding region operatively linked to a DNA segment encoding thecoagulation factor and expressing the vector in a recombinant host cellso that it produces the encoded fusion protein.

Coagulation factors for use in the invention may comprise one of thevitamin K-dependent coagulant factors, such as Factor II/IIa, FactorVII/VIIa, Factor IX/IXa or Factor X/Xa. Factor V/Va, VIII/VIIIa, FactorXI/XIa, Factor XII/XIIa and Factor XIII/XIIIa may also be used.

Particular aspects concern the vitamin K-dependent coagulation factorsthat lack the Gla modification. Such factors may be prepared byexpressing a vitamin K-dependent coagulation factor-encoding gene in aprocaryotic host cell (which cells are unable to effect the Glu to Glamodification). The factors may also be prepared by making an engineeredcoagulation factor gene that encodes a vitamin K-dependent coagulationfactor lacking the necessary or "corresponding" Glutamic acid residues,and then expressing the engineered gene in virtually any recombinanthost cell. Equally, such a coagulation factor may be prepared bytreating the vitamin K-dependent coagulation factor protein to remove oralter the corresponding Glutamic acid residues.

Preferred coagulation factors for use in the binding ligands of theinvention are Tissue Factor and Tissue Factor derivatives. One group ofuseful Tissue Factors are those mutants deficient in the ability toactivate Factor VII. A Tissue Factor may be rendered deficient in theability to activate Factor VII by altering one or more amino acids fromthe region generally between about position 157 and about position 167in the amino acid sequence. Exemplary mutants are those wherein Trp atposition 158 is changed to Arg; wherein Ser at position 162 is changedto Ala; wherein Gly at position 164 is changed to Ala; and the doublemutant wherein Trp at position 158 is changed to Arg and Ser at position162 is changed to Ala.

Further preferred Tissue Factor derivatives are truncated TissueFactors, dimeric or even polymeric Tissue Factors and dimeric, or evenpolymeric, truncated Tissue Factors.

The present invention further provides novel Tissue Factor constructsthat comprise a Tissue Factor or derivative operatively linked to atleast one other Tissue Factor or derivative. Truncated Tissue Factorsare preferred, with truncated Tissue Factors that have been modified tocomprise a hydrophobic membrane insertion moiety being particularlypreferred.

"A hydrophobic membrane insertion moiety", as defined herein, is one ormore units that direct the insertion or functional contact of the TissueFactor with a membrane. The hydrophobic membrane insertion moieties ofthe invention are exemplified by stretches of substantially hydrophobicamino acids, such as between about 3 and about 20 hydrophobic aminoacids; and also by fatty acids.

The hydrophobic amino acids may be located either at the N- orC-terminus of the truncated Tissue Factor, or appended at another pointof the molecule. Where hydrophobic amino acids are used, they may beadvantageously incorporated into the molecule by molecular biologicaltechniques. Equally, hydrophobic amino acids or fatty acids may be addedto the Tissue Factor using synthetic chemistry techniques.

In the Tissue Factor dimers, trimers and polymers of the presentinvention, each of the Tissue Factors or derivatives may be operativelylinked via, e.g., a disulfide, thioether or peptide bond. In certainembodiments, the Tissue Factor units will be linked via a bond that issubstantially stable in plasma, or in the physiological environment inwhich it is intended for use. This is based upon the inventors' conceptthat the dimeric form of Tissue Factor may prove to be the mostbiologically active. However, there is no requirement for a stablelinkage as Tissue Factor monomers are known to be active in the methodsof the invention.

One or more of the Tissue Factors or truncated Tissue Factors in thedimers and multimers may also be modified to contain a terminal cysteineresidue or another moiety that is suitable for linking the Tissue Factorconstruct to a second agent, such as a binding region.

Tissue Factor monomers, truncated Tissue Factors, and Tissue Factordimers and multimers that contain a peptide that includes aselectively-cleavable amino acid sequence therefore form another aspectof the invention. Peptide linkers that include a cleavage site forurokinase, plasmin, Thrombin, Factor IXa, Factor Xa or ametalloproteinase, such as an interstitial collagenase, a gelatinase ora stromelysin, are particularly preferred.

The Tissue Factor monomers, truncated Tissue Factors, Tissue Factordimers and multimers, and indeed any coagulant, may therefore be linkedto a second agent, such as an antibody, an antigen binding region of anantibody, a ligand or a receptor, via a biologically-releasable bond.The preference for peptide linkers that include a cleavage site for theabove listed proteinases is based on the presence of such proteinaseswithin, e.g., a tumor environment. The delivery of a bispecific agent orligand to the tumor site is expected to result in cleavage, resulting inthe relatively specific release of the coagulation factor.

Particular constructs of the invention are those comprising anoperatively linked series of units in the sequence: a cysteine residue,a selectively cleavable peptide linker, a stretch of hydrophobic aminoacids, a first truncated Tissue Factor and a second truncated TissueFactor; or in the sequence: a first cysteine residue, a selectivelycleavable peptide linker, a first stretch of hydrophobic amino acids, afirst truncated Tissue Factor, a second truncated Tissue Factor and asecond stretch of hydrophobic amino acids; wherein each construct may ornot be linked to a second agent such as an antibody, ligand or receptor.

Other suitable coagulation factors are Russell's viper venom Factor Xactivator; platelet-activating compounds, such as thromboxane A₂ andthromboxane A₂ synthase; and inhibitors of fibrinolysis, such asα2-antiplasmin.

Also encompassed by the invention are binding ligands in which thecoagulation factor is not covalently linked to the conjugate, but isnon-covalently bound thereto by means of binding to a second bindingregion that is operatively linked to the targeting agent of theconstruct. Suitable "second binding regions" include antigen combiningsites of antibodies that have binding specificity for the coagulationfactor, including functional portions of antibodies, such as scFv, Fv,Fab', Fab and F(ab')₂ fragments.

Binding ligands that contain antibodies, or fragments thereof, directedagainst the vitamin K-dependent coagulant Factor II/IIa, FactorVII/VIIa, Factor IX/IXa or Factor X/Xa; a vitamin K-dependentcoagulation factor that lacks the Gla modification; Tissue Factor, amutant Tissue Factor, a truncated Tissue Factor, a dimeric TissueFactor, a polymeric Tissue Factor, a dimeric truncated Tissue Factor;Prekallikein; Factor v/va, VIII/VIIIa, Factor XI/XIa, Factor XII/XIIa,Factor XIII/XIIIa; Russell's viper venom Factor X activator, thromboxaneA₂ or α2-antiplasmin are therefore contemplated.

The non-covalently bound coagulating agents may be bound to, or"precomplexed", with a coagulation factor, e.g., so that they may beused to deliver an exogenous coagulation factor to a disease site, e.g.,the tumor vasculature, of an animal upon administration. Equally,binding ligands that comprise a second binding region that is specificfor a coagulation factor may also be administered to an animal in an"uncomplexed" form and still function to achieve specific coagulation;in which instance, the agent would garner circulating (endogenous)coagulation factor and concentrate it within the disease or tumor site.

In terms of the "coagulation factors" or coagulating agents, these maybe endogenous coagulation factors and derivatives thereof, orexogenously added version of such factors, including recombinantversions. Coagulants (in the present "coaguligands") have the distinctadvantage over toxins (in immunotoxins) as they will not producesignificant adverse side effects upon targeting to a marker that provesto be less than 100% disease-restricted. Furthermore, the coagulantsused will most often be of human origin, and will therefore pose lessimmunogenicity problems than foreign toxins, such as ricin A chain.

Although not limited to such compositions, important examples ofcompositions in accordance with this invention are bispecificantibodies, which antibodies comprise a first antigen binding regionthat binds to a disease cell or component of disease-associatedvasculature marker and a second antigen binding region that binds to acoagulation factor. The invention also provides scFv, Fv, Fab', Fab andF(ab')₂ fragments of such bispecific antibodies. One currently preferredexample of such a bispecific antibody is an antibody comprising onebinding site directed against an MHC Class II antigen and anotherbinding site directed against Tissue Factor.

In further embodiments, the present invention provides pharmaceuticalcompositions of, and therapeutic kits comprising, any or a combinationof the above binding ligands and bispecific antibodies inpharmacologically acceptable forms. This includes pharmaceuticalcompositions and kits where the binding ligand has a first bindingregion that is covalently linked to a coagulation factor, and alsobinding ligands in which the first binding region is covalently linkedto a second binding region that, in turn, binds to the coagulationfactor--whether binding occurs prior to, or subsequent to,administration to an animal.

Pharmaceutical compositions and therapeutic kits that include acombination of bispecific, trispecific or multispecific binding ligandsin accordance with the invention are also contemplated. This includescombinations where one binding ligand is directed against a diseasedcell or a tumor cell and where another is directed against a vasculatureendothelial cell marker or component of disease-associated stroma. Otherdistinct components may also be included in the compositions and kits ofthe invention, such as antibodies, immunotoxins, immunoeffectors,chemotherapeutic agents, and the like.

The kits may also include an antigen suppressor, such as a cyclosporin,for use in suppressing antigen expression in endothelial cells of normaltissues; and/or an "inducing agent" for use in inducingdisease-associated vascular endothelial cells or stroma to express atargetable antigen, such as E-selectin, P-selectin or an MHC Class IIantigen. Exemplary inducing agents include T cell clones that binddisease or tumor antigens and that produce IFN-γ, although it iscurrently preferred that such clones be isolated from the animal to betreated using the kit.

Preferred inducing agents are bispecific antibodies that bind to diseaseor tumor cell antigens, or even stromal components, and to effectorcells capable of producing cytokines, coagulants, or other factors, thatinduce expression of desired target antigens. Currently, one preferredgroup of bispecific antibodies are those that bind to a tumor antigenand to the activation antigens CD14 or CD16, to stimulate IL-1production by monocytes, macrophages or mast cells; and those that bindto a tumor antigen and to the activation antigens CD2, CD3 or CD28, andpreferably CD28, to stimulate IFN-γ production by NK cells or preferablyby T cells.

A second preferred group of bispecific antibodies are those that bind toa tumor antigen or to a component of tumor stroma, and to Tissue Factor,a Tissue Factor derivative, prothrombin, Factor VII/VIIa, Factor IX/IXa,Factor X/Xa, Factor XI/XIa or Russell's viper venom Factor X activator,to stimulate thrombin production. Kits comprising such bispecificantibodies as a first "inducing" composition will generally include asecond pharmaceutical composition that comprises a binding ligand thatcomprises a first binding region that binds to P-selectin or E-selectin.

The bispecific ligands of the invention, and other components asdesired, may be conveniently aliquoted and packaged, using one or moresuitable container means, and the separate containers dispensed in asingle package. Pharmaceutical compositions and kits are furtherdescribed herein.

Although the present invention has significant clinical utility in thedelivery of coagulants and in disease treatment, it also has many invitro uses. These include, for example, various assays based upon thebinding ability of the particular antibody, ligand or receptor, of thebispecific compounds. The bispecific coagulating ligands of inventionmay thus be employed in standard binding assays and protocols, such asin immunoblots, Western blots, dot blots, RIAS, ELISAs,immunohistochemistry, fluorescent activated cell sorting (FACS),immunoprecipitation, affinity chromatography, and the like, as furtherdescribed herein.

In still further embodiments, the invention concerns methods fordelivering a coagulant to disease-associated vasculature, as may be usedto treat diseases such as diabetic retinopathy, vascular restenosis,AVM, hemangioma, neovascular glaucoma, psoriasis and rheumatoidarthritis, and tumors that have a vascularized tumor component. Suchmethods generally comprise administering to an animal, including a humansubject, with a disease that has a vascular component, a pharmaceuticalcomposition comprising at least one bispecific binding ligand inaccordance with those described above.

The compositions are administered in amounts and by routes effective topromote blood coagulation in the vasculature of the disease site, e.g.,a solid tumor. Effective doses will be known to those of skill in theart in light of the present disclosure, such as the information in thePreferred Embodiments and Detailed Examples. Parenteral administrationwill often be suitable, as will other methods, such as, e.g., injectioninto a vascularized tumor site.

The methods of the invention provide for the delivery of exogenouscoagulation factors, by means of both administering a binding ligandthat comprises a covalently-bound coagulation factor and by means ofadministering a binding ligand that comprises a non-covalently boundcoagulation factor that is complexed to a second binding region of thebispecific ligand or antibody.

Further methods of the invention include those that result in thedelivery of an endogenous coagulation factor to disease or tumorvasculature. This is achieved by administering to the animal or patienta binding ligand that comprises a second binding region that binds toendogenous coagulation factor and concentrates the factor at thedisease-associated or tumor vasculature.

In yet still further methodological embodiments, it is contemplated thatmarkers of tumor vasculature or stroma may be specifically induced andthen targeted using a binding ligand, such as an antibody. Exemplaryinducible antigens include E-selectin, P-selectin, MHC Class IIantigens, VCAM-1, ICAM-1, endoglin, ligands reactive with LAM-1,vascular addressins and other adhesion molecules, with E-selectin andMHC Class II antigens being currently preferred.

When inducing and subsequently targeting MHC Class II proteins, thesuppression of MHC Class II in normal tissues is generally required. MHCClass II suppression may be achieved using a cyclosporin, or afunctionally equivalent agent. MHC Class II molecules may then beinduced in disease-associated vascular endothelial cells usingcyclosporin-independent means, such as by exposing thedisease-associated vasculature to an effector cell, generally a Helper Tcell or NK cell, of the animal that releases the inducing cytokineIFN-γ.

Activated monocytes, macrophages and even mast cells are effector cellscapable of producing cytokines (IL-1; TNF-α; TNF-β) that induceE-selectin; whereas Helper T cells, CD8-positive T cells and NK cellsare capable of producing IFN-γ that induces MHC Class II. Activatingmonocyte/macrophages in the disease site to produce IL-1, or activatingdisease-associated Helper T cells or NK cells to produce IFN-γ, may beachieved by administering to the animal an activating antibody thatbinds to an effector cell surface activating antigen. Exemplaryactivating antigens include CD14 and CD16 (FcR for IgE) formonocytes/macrophages; and CD2, CD3 and CD28 for T cells; with CD14 andCD28, respectively, being preferred for use in certain embodiments.

To achieve specific activation and induction, one currently preferredmethod is to use a bispecific antibody that binds to both an effectorcell activating antigen, such as CD14 or CD28, and to a disease or tumorcell antigen. These bispecific antibodies will localize to the diseaseor tumor site and activate monocyte/macrophages and T cells,respectively. The activated effector cells in the vicinity of thetargeted disease or tumor component will produce inducing cytokines, inthis case, IL-1 and IFN-γ, respectively.

MHC Class II suppression in normal tissues may also be achieved byadministering to an animal an anti-CD4 antibody; this functions tosuppress IFN-γ production by T cells of the animal resulting ininhibition of MHC Class II expression. MHC Class II molecules may againbe specifically induced in disease-associated vascular endothelial cellsby exposing only the disease site to IFN-γ. One means by which toachieve this is by administering to the animal an IFN-γ-producing T cellclone that binds to an antigen in the disease site. The IFN-γ-producingT cells will preferably be infiltrating leukocytes obtained from thedisease site of the animal, such as tumor infiltrating leukocytes (TILs)expanded in vitro.

Methods using bispecific antibodies to induce coagulant, such asthrombin, production, only in a local environment, such as in a tumorsite, are also provided. Again, this will generally be achieved byadministering to an animal a pharmaceutical composition comprising abispecific antibody that binds to a tumor cell or a component of tumorstroma and to Tissue Factor, a Tissue Factor derivative, prothrombin,Factor VII/VIIa, Factor IX/IXa, Factor X/Xa, Factor XI/XIa or Russell'sviper venom Factor X activator. Antibodies that bind to E-selectin orP-selectin are then linked to a coagulation factor or a second bindingregion that binds to a coagulation factor and are introduced into thebloodstream of an animal.

More conventional combination treatment regimens are also possiblewhere, for example, a tumor coagulating element of this invention iscombined with an existing antitumor therapy, such as with radiotherapyor chemotherapy, or through the use of a second immunological reagent,such as an antitumor immunotoxin. The novel treatment methods for benigndiseases can also be combined with other presently used therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Tethering of tTF to A20 cells via B21-2/10H10 bispecificantibody. A20 cells were incubated with varying concentrations ofB21-2/10H10 (□), SFR8/10H10 () or B21-2/OX7 (∘) plus an excess of ¹²⁵I-tTF for 1 h at 4° C. in the presence of sodium azide. The number of¹²⁵ I-tTF associated with the cells was determined as described inExample II.

FIG. 2. Relationship between number of tethered tTF molecules per A20cell and ability to induce coagulation of plasma. A20 cells wereincubated with varying concentrations of B21-2/10H10 plus an excess oftTF for 1 h at 4° C. in the presence of sodium azide. The cells werewashed, warmed to 37° C., calcium and mouse plasma were added and thetime for the first fibrin strands to form was recorded (abscissa). Anidentical study was performed in which the A20 cells were incubated for1 h at 4° C. with bispecific antibody plus ¹²⁵ I-tTF and the number oftTF specifically bound to the cells was determined as described inExample II (ordinate). Plasma added to untreated A20 cells (i.e. zerotTF molecules/cell) coagulated in 190 seconds.

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D. Time course of vascularthrombosis and tumor necrosis after administration of coaguligand.Groups of 3 mice bearing 0.8 cm diameter C1300 (Muγ) tumors were givenan intravenous injection of a coaguligand composed of 14 μg B21-2/10H10and 11 μg tTF. FIG. 3A; Before injection: blood vessels are intact andtumor cells are healthy. FIG. 3B; 0.5 hours: blood vessels throughoutthe tumor are thrombosed; tumor cells are healthy. FIG. 3C; 4 hours:dense thrombi are present in all tumor vessels and tumor cells areseparating and developing pyknotic nuclei. Erythrocytes are visible inthe tumor interstitium. FIG. 3D; 24 hours: advanced tumor necrosisthroughout the tumor. Arrows indicate blood vessels.

FIG. 4. Solid tumor regression induced by tumor-vasculature directedcoaguligand therapy. Nu/nu mice bearing approximately 0.8 cm diameterC1300 (Muγ) tumors were given two intravenous injections of B21-2/10H10(14 μg) mixed with tTF (11 μg) spaced 1 week apart (arrows) (□). Mice incontrol groups received equivalent doses of tTF alone (), B21-2/10H10alone (∘) or diluent (▪). Other control groups which received equivalentdoses of isotype-matched control bispecific antibodies (SFR8/10H10,OX7/10H10 or B21-2/OX7) and tTF had similar tumor responses to those inanimals receiving tTF alone. The number of mice per group was 7 or 8.

FIG. 5. Exemplary antibody-tTF constructs. This figure shows both theconjugates synthesized by the linkage of chemically derivatized antibodyto chemically derivatized tTF via a disulfide bond, and also the linkageof various TF or TF dimers to antibodies and fragments thereof.

FIG. 6. Clotting activity of tTF conjugates when bound to A20 cells. A20cells were incubated with varying concentrations of B21-2/10H10bispecific+H₆ [tTF] in a 1:1 molar ratio, premixed for one hour (□),B21-2 antibody-H₆ C[tTF] (), and B21-2 antibody-H₆ [tTF] (▴) for 1 hourat 4° C. in the presence of sodium azide. The cells were washed, warmedto 37° C., calcium and mouse plasma were added and the time for thefirst fibrin strands to form was recorded. The results are expressed asclotting time as a % of the clotting time in the absence of tTF.

FIG. 7. Clotting activity of anti-tumor cell tTF conjugates. LS174Tcells (▪), Widr cells () and H460 cells (▴), preincubated with TF9-6B4and TF8-5G9 antibodies, were incubated with varying concentrations ofD612 antibody-H₆ C[tTF] (▪), KS1/4 antibody-H₆ [tTF] (), and XMMCO791antibody-H₆ [tTF] (▴) for 1 hour at 4° C. in the presence of sodiumazide. The cells were washed, warmed to 37° C., calcium and mouse plasmawere added and the time for the first fibrin strands to form wasrecorded. The results are expressed as clotting time as a % of theclotting time in the absence of tTF.

FIG. 8. Gla domains (γ-carboxyglutamic acid) of Factor II/IIa, FactorVII/IIa, Factor IX/IXa and Factor X/Xa. The arrows represent signalpeptide and pro-peptide cleavage sites and activating cleavage sites(slanted arrows).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although they show great promise in the therapy of lymphomas andleukemias (Lowder et al., 1987; Vitetta et al., 1991), monoclonalantibodies (MAbs) and immunotoxins (ITs) have thus far proved relativelyineffective in clinical trials against carcinomas and other solid tumors(Byers & Baldwin, 1988; Abrams & Oldham, 1985), which account for morethan 90% of all cancers in man (Shockley et al., 1991). A principalreason for this is that macromolecules do not readily extravasate intosolid tumors (Sands, 1988; Epenetos et al., 1986) and, once within thetumor mass, fail to distribute evenly due to the presence of tightjunctions between tumor cells (Dvorak et al., 1991), fibrous stroma(Baxter et al., 1991), interstitial pressure gradients (Jain, 1990) andbinding site barriers (Juweid et al., 1992).

In developing new strategies for treating solid tumors, the methods thatinvolve targeting the vasculature of the tumor, rather than the tumorcells themselves, therefore seem to offer certain advantages. Inducing ablockade of the blood flow through the tumor, e.g., through tumorvasculature specific fibrin formation, would interfere with the influxand efflux processes in a tumor site, thus resulting in anti-tumoreffect. Arresting the blood supply to a tumor may be accomplishedthrough shifting the procoagulant-fibrinolytic balance in thetumor-associated vessels in favour of the coagulating processes byspecific exposure to coagulating agents.

The present invention provides various means for effecting specificblood coagulation, as exemplified by tumor-specific coagulation. This isachieved using bispecific or multispecific binding ligands in which atleast one component is an immunological- or growth factor-basedtargeting component, and at least one other component is provided thatis capable of directly, or indirectly, stimulating coagulation.

A. Targetable Disease Sites

The compositions and methods provided by this invention are broadlyapplicable to the treatment of any disease, such as a benign ormalignant tumor, having a vascular component. Suchvasculature-associated diseases include BPH, diabetic retinopathy,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, neovascular glaucoma and psoriasis; and also angiofibroma,arthritis, atherosclerotic plaques, corneal graft neovascularization,hemophilic joints, hypertrophic scars, osler-weber syndrome, pyogenicgranuloma retrolental fibroplasia, scleroderma, trachoma, vascularadhesions, synovitis, dermatitis and even endometriosis.

Typical vascularized tumors are the solid tumors, particularlycarcinomas, which require a vascular component for the provision ofoxygen and nutrients. Exemplary solid tumors that may be treated usingthe invention include, but are not limited to, carcinomas of the lung,breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, prostate, thyroid, squamous cell carcinomas,adenocarcinomas, small cell carcinomas, melanomas, gliomas,neuroblastomas, and the like.

One binding region of the bispecific agents of the invention will be acomponent that is capable of delivering the coagulating agent to thetumor region, i.e., capable of localizing within a tumor site, such asthose described above. As somewhat wider distribution of the coagulatingagent will not be associated with severe side effects, such as is knownto occur with a toxin moiety, there is a less stringent requirementimposed on the targeting element of the bispecific ligand. The targetingagent may thus be directed to components of tumor cells; components oftumor vasculature; components that bind to, or are generally associatedwith, tumor cells; components that bind to, or are generally associatedwith, tumor vasculature; components of the tumor extracellular matrix orstroma; and even cell types found within the tumor vasculature.

The burden of very stringent targeting, e.g., as imposed when usingimmunotoxins, is also lessened due to the fact that tumor vasculature is`prothrombotic` and is predisposed towards coagulation. Therefore, toachieve specific targeting means that coagulation is promoted in thetumor vasculature relative to the vasculature in non-tumor sites. Thus,specific targeting is a functional term rather than a purely physicalterm relating to the biodistribution properties of the targeting agent,and it is not unlikely that useful targets may be not be entirelytumor-restricted, and that targeting ligands which are effective topromote tumor-specific coagulation may nevertheless be found at othersites of the body following administration.

1. Tumor Cell Targets

The malignant cells that make up the tumor may be targeted using abispecific ligand that has a region capable of binding to a relativelyspecific marker of the tumor cell. In that binding to tumor cells willlocalize the associated coagulating agent to the tumor, specificcoagulation will be achieved. Furthermore, it is expected that thiswould be a particularly effective means of promoting coagulation as, dueto the physical accessibility of perivascular tumor cells, thebispecific agents will likely be concentrated around the tumor cellsthat are nearest to a blood vessel.

Many so-called "tumor antigens" have been described, any one which couldbe employed as a target in connection with the present invention. Alarge number of exemplary solid tumor-associated antigens are listedherein in Table I. The preparation and use of antibodies against suchantigens is well within the skill of the art, and exemplary antibodiesare also listed in Table I.

Another means of defining a targetable tumor is in terms of thecharacteristics of a tumor cell itself, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,Table II is provided for the purpose of exemplifying human tumor celllines that are publically available (from ATCC Catalogue).

The information presented in Table II is by means of an example, and notintended to be limiting either by year or by scope. One may consult theATCC Catalogue of any subsequent year to identify other appropriate celllines. Also, if a particular cell type is desired, the means forobtaining such cells, and/or their instantly available source, will beknown to those of skill in the particular art. An analysis of thescientific literature will thus readily reveal an appropriate choice ofcell for any tumor cell type desired to be targeted.

                                      TABLE I                                     __________________________________________________________________________    MARKER ANTIGENS OF SOLID TUMORS AND CORRESPONDING MONOCLONAL ANTIBODIES                   Antigen                                                                        Identity/                                                                                Monoclonal                                            Tumor Site   Characteristics                                                                         Antibodies                                                                               Reference                                   __________________________________________________________________________    A: Gynecological                                                                          `CA 125` >200                                                                           OC 125     Kabawat et al., 1983; Szymendera, 1986       GY           kD mucin GP                                                      ovarian     80 Kd GP  OC 133     Masuko et al, Cancer Res., 1984              ovarian     `SGA` 360 Kd GP                                                                         OMI         de Krester et al., 1986                     ovarian     High M.sub.r mucin                                                                        Mo v1     Miotti et al, Cancer Res., 1985             ovarian     High M.sub.r mucin/                                                                      Mo v2      Miotti et al, Cancer Res., 1985                           glycolipid                                                      ovarian     NS              3C2     Tsuji et al., Cancer Res., 1985           ovarian     NS              4C7     Tsuji et al., Cancer Res., 1985           ovarian     High M.sub.r mucin                                                                        ID.sub.3   Gangopadhyay et al., 1985                  ovarian     High M.sub.r mucin                                                                        DU-PAN-2   Lan et al., 1985                           GY               7700 Kd GP                                                                              F 36/22                                                                                   Croghan et al., 1984                   ovarian     `gp 68` 48 Kd GP                                                                          4F.sub.7 /7A.sub.10                                                                      Bhattacharya et a1., 1984                  GY               40, 42kD GP                                                                            OV-TL3 Poels et al., 1986                           GY               `TAG-72` High                                                                        B72.3     Thor et al., 1986                                        M.sub.r mucin                                                    ovarian     300-400 Kd GP                                                                             DF.sub.3    Kufe et al., 1984                         ovarian     60 Kd GP  2C.sub.8 /2F.sub.7                                                                          Bhattacharya et al., 1985                 GY               105 Kd GP                                                                             MF 116  Mattes et al., 1984                          ovarian     38-40 kD GP                                                                              MOv18      Miotti et al., 1987                         GY           `CEA` 180 Kd GP                                                                         CEA 11-H5  Wagener et al., 1984                        ovarian       CA 19-9 or GICA                                                                         CA 19-9 (1116NS 19-9)                                                                  Atkinson et al., 1982                        ovarian           `PLAP` 67 Kd GP                                                                    H17-E2    McDicken et al., 1985                        ovarian           72 Kd                                                                                  791T/36                                                                                   Perkins et al., 1985                   ovarian           69 Kd PLAP                                                                        NDOG.sub.2  Sunderland et al., 1984                     ovarian           unknown M.sub.r PLAP                                                                H317       Johnson et al., 1981                       ovarian           p185.sup.HER2                                                                      4D5, 3H4, 7C2, 6E9,                                                                      Shepard et al., 1991                                                2C4, 7F3, 2H11,                                                               3E8, 5B8, 7D3, SB8                                    uterus ovary                                                                               HMFG-2     HMFG2     Epenetos et al.,  1982                      GY             HMFG-2   3.14.A3      Burchell et al.,  1983                   B: BREAST     330-450 Kd GP                                                                           DF3        Hayes et al., 1985                                     NS         NCRC-11         Ellis et al., 1984                                 37kD        3C6F9     Mandeville et al., 1987                                 NS         MBE6        Teramoto et al.,  1982                                 NS         CLNH5      Glassy et al., 1983                                     47 Kd GP    MAC 40/43                                                                                  Kjeldsen et al., 1986                                High M.sub.r GP                                                                         EMA           Sloane et al., 1981                                   High M.sub.r GP                                                                         HMFG1 HFMG2                                                                                Arklie et al., 1981                                    NS         3.15.C3         Arklie et al., 1981                                NS         M3, M8, M24                                                                               Foster et al., 1982                                    1 (Ma) blood                                                                                 M18      Foster et al., 1984                                   group Ags                                                                     NS          67-D-11        Rasmussen et al., 1982                             oestrogen   D547Sp, D75P3, H222                                                                     Kinsel et al., 1989                                     receptor                                                                      EGF Receptor                                                                                  Anti-EGF                                                                                Sainsbury et al., 1985                              Laminin       LR-3     Horan Hand et al., 1985                                Receptor                                                                      erb B-2 p185                                                                                  TA1     Gusterson et al., 1988                                NS          H59         Hendler et al., 1981                                  126 Kd GP   10-3D-2        Soule et al., 1983                                 NS          HmAB1,2        Imam et al., 1984; Schlom et al.,                                       1985                                                     NS          MBR 1,2,3                                                                                  Menard et al., 1983                                  95 Kd           24.17.1                                                                              Thompson et al., 1983                                  100 Kd         24.17.2 (3E1.2)                                                                        Croghan et al., 1983                                  NS          F36/22.M7/105                                                                          Croghan et al., 1984                                     24 Kd           C11, G3, H7                                                                          Adams et al., 1983                                     90 Kd GP     B6.2      Colcher et al., 1981                                   CEA & 180 Kd GP                                                                            B1.1      Colcher et al., 1983                                   colonic &   Cam 17.1      Imperial Cancer Research Technology                                      MAb                                                      pancreatic                                                                                          listing                                                 mucin similar                                                                 to Ca 19-9                                                                    milk mucin core                                                                          SM3          Imperial Cancer Research Technology                                        Mab                                                      protein                listing                                                milk mucin core                                                                          SM4           Imperial Cancer Research Technology                                       Mab                                                      protein                listing                                                affinity- C-Mul (566)                                                                               Imperial Cancer Research Technology                                          Mab                                                      purified milk                                                                                       listing                                                 mucin                                                                         p185.sup.HER2                                                                            4D5 3H4, 7C2, 6E9,                                                                        Shepard et al., 1991                                                 2C4, 7F3, 2H11,                                                              3E8, 5B8, 7D3, 5B8                                               CA 125 >200 Kd                                                                            OC 125    Kabawat et al., 1985                                    GP                                                                            High M.sub.r mucin/                                                                      MO v2       Miotti et al., 1985                                    glycolipid                                                                    High M.sub.r mucin                                                                         DU-PAN-2                                                                                    Lan et al., 1984                                   `gp48` 48 Kd GP                                                                          4F.sub.7 /7A.sub.10                                                                      Bhattacharya et al., 1984                               300-400 Kd GP                                                                              DF.sub.3                                                                                  Kufe et al., 1984                                    `TAG-72` high                                                                              B72.3     Thor et al., 1986                                      M.sub.r mucin                                                                 `CEA` 180 Kd GP                                                                         cccccCEA 11                                                                                 Wagener et al., 1984                                  `PLAP`  67 Kd GP                                                                         H17-E2     McDicken et al., 1985                                   HMFG-2 >400 Kd GP                                                                         3.14.A3  Burchell et al., 1983                                    NS        FO23C5      Riva et al., 1988                           C: COLORECTAL                                                                             TAG-72 High M.sub.r                                                                     B72.3        Colcher et al., 1987                                    mucin                                                                         GP37        (17-1A) 1083-17-1A                                                                      Paul et al., 1986                                       Surface GP                                                                                   CO17-1A                                                                             LoBuglio et al., 1988                                   CEA       ZCE-025          Patt et al., 1988                                  CEA       AB2           Griffin et al., 1988a                                 cell surface AG                                                                           HT-29-15      Cohn et al., 1987                                   secretory  250-30.6       Leydem et al., 1986                                 epithelium                                                                    surface      44X14    Gallagher et al., 1986                                  glycoprotein                                                                  NS         A7            Takahashi et al., 1988                               NS         GA73.3    Munz et al., 1986                                        NS         791T/36         Farrans et al., 1982                               cell membrane &                                                                         28A32       Smith et al., 1987                                      cytoplasmic Ag                                                                CEA & vindesine                                                                           28.19.8        Corvalen, 1987                                     gp72            X MMCO-791                                                                            Byers et al., 1987                                    high M.sub.r mucin                                                                          DU-PAN-2                                                                                  Lan et al., 1985                                    high M.sub.r mucin                                                                         ID.sub.3                                                                                 Gangopadhyay et al., 1985                             CEA 180 Kd GP                                                                               CEA 11-H5                                                                                Wagener et al., 1984                                 60 Kd GP    2C.sub.8 /2F.sub.7                                                                           Bhattacharya et al., 1985                          CA-19-9 (or                                                                                  CA-19-9 (1116NS 19-9)                                                               Atkinson et al., 1982                                    GICA)                                                                         Lewis a      PR5C5    Imperial Cancer Research Technology                                          Mab                                                                           Listing                                                  Lewis a      PR4D2    Imperial Cancer Research Technology                                          Mab                                                                           Listing                                                  colonic mucus                                                                               PR4D1     Imperial Cancer Research Technology                                        Mab                                                                           Listing                                      D: MELANOMA  p97.sup.a                                                                                    4.1       Woodbury et al., 1980                               p97.sup.a     8.2 M.sub.17                                                                          Brown, et al.,  1981a                                   p97.sup.b     96.5       Brown, et al.,  1981a                                p97.sup.c     118.1, 133.2,                                                                         Brown, et al.,  1981a                                             (113.2)                                                             p97.sup.c  L.sub.1, L.sub.10, R.sub.10 (R.sub.19)                                                    Brown et al., 1981b                                    p97.sup.d   I.sub.12   Brown et al.,  1981b                                   p97.sup.e    K.sub.5   Brown et al.,  1981b                                   p155            6.1     Loop et al.,  1981                                    G.sub.D3 disialogan-                                                                      R24         Dippold et al.,  1980                                 glioside                                                                      p210, p60, p250                                                                           5.1           Loop et al.,  1981                                  p280 p440  225.28S    Wilson et al.,  1981                                    GP 94, 75, 70 & 25                                                                      465.12S     Wilson et al.,  1981                                    P240-P250, P450                                                                           9.2.27     Reisfeld et al., 1982                                  100, 77, 75 Kd                                                                           F11         Chee et al.,  1982                                     94 Kd        376.96S  Imai et al., 1982                                       4 GP chains                                                                              465.12S    Imai et al., 1982; Wilson et al., 1981                  GP 74          15.75    Johnson & Reithmuller, 1982                           GP 49          15.95    Johnson & Reithmuller,  1982                          230 Kd        Mel-14   Carrel et al.,  1982                                   92 Kd       Mel-12   Carrel et al., 1982                                      70 Kd       Me3-TB7        Carrel et al., 1:387, 1982                         HMW MAA similar                                                                           225.28SD   Kantor et al., 1982                                    to 9.2.27 AG                                                                  HMW MAA similar                                                                           763.24TS   Kantor et al., 1982                                    to 9.2.27 AG                                                                  GP95 similar to                                                                           705F6     Stuhlmiller et al,, 1982                                376.96S 465.12S                                                               GP125           436910                                                                             Saxton et al., 1982                                      CD41      M148         Imperial Cancer Research Technology                                         Mab                                                                             listing                                    E: GASTROINTESTINAL                                                                       high M.sub.r mucin                                                                       ID3        Gangopadhyay et al., 1985                   pancreas, stomach                                                             gall bladder,                                                                                high M.sub.r mucin                                                                       DU-PAN-2                                                                               Lan et al., 1985                           pancreas, stomach                                                             pancreas        NS      OV-TL3   Poels et al., 1984                           pancreas, stomach,                                                                         `TAG-72` high                                                                           B72.3      Thor et al., 1986                           oesophagus    M.sub.r mucin                                                   stomach          `CEA` 180 Kd GP                                                                      CEA 11-H5                                                                                  Wagener et al., 1984                     pancreas        HMFG-2 >400 Kd GP                                                                       3.14.A3                                                                                 Burchell et al., 1983                     G.I.         NS         C COLI   Lemkin et al., 1984                          pancreas, stomach                                                                           CA 19-9 (or                                                                            CA-19-9 (1116NS 19-9)                                                                   Szymendera, 1986                                          GICA)        and CA50                                            pancreas        CA125 GP                                                                             OC125      Szymendera, 1986                            F: LUNG          p185.sup.HER2                                                                       4D5 3H4, 7C2, 6E9,                                                                        Shepard et al., 1991                       non-small cell        2C4, 7F3, 2H11,                                         lung carcinoma                                                                                        3E8, 5B8, 7D3, SB8                                                high M.sub.r mucin/                                                                       MO v2     Miotti et al., 1985                                     glycolipid                                                                    `TAG-72` high                                                                             B72.3     Thor et al., 1986                                       M.sub.r mucin                                                                 high M.sub.r mucin                                                                         DU-PAN-2                                                                               Lan et al., 1985                                        `CEA` 180 kD GP                                                                           CEA 11-H5                                                                               Wagener et al., 1984                        Malignant Gliomas                                                                          cytoplasmic                                                                              MUC 8-22   Stavrou, 1990                                          antigen from                                                                  85HG-22 cells                                                                 cell surface Ag                                                                           MUC 2-63      Stavrou, 1990                                       from 85HG-63                                                                  cells                                                                         cell surface Ag                                                                           MUC 2-39      Stavrou, 1990                                       from 86HG-39                                                                  cells                                                                         cell surface Ag                                                                           MUC 7-39      Stavrou, 1990                                       from 86HG-39                                                                  cells                                                             G: MISCELLANEOUS                                                                           p53      PAb 240     Imperial Cancer Research Technology                                          MaB                                                                PAb 246          Listing                                                       PAb 1801                                               small round cell                                                                           neural cell                                                                             ERIC.1     Imperial Cancer Research Technology                                          MaB                                          tumors         adhesion                                                                                          Listing                                                 molecule                                                         medulloblastoma        M148       Imperial Cancer Research Technology                                          MaB                                          neuroblastoma                                                                                                    Listing                                    rhabdomyosarcoma                                                              neuroblastoma           FMH25    Imperial Cancer Research Technology MaB                                       Listing                                      renal cancer &                                                                             p155       6.1        Loop et al., 1981                          glioblastomas                                                                 bladder &    "Ca Antigen"                                                                             CA1       Ashall et al., 1982                         laryngeal cancers                                                                            350-390 kD                                                     neuroblastoma                                                                             GD2       3F8          Cheung et al., 1986                        Prostate      gp48 48 kD GP                                                                           4F.sub.7 /7A.sub.10                                                                      Bhattacharya et al., 1984                  Prostate      60 kD GP                                                                                2C.sub.8 /2F.sub.7                                                                       Bhattacharya et al., 1985                  Thyroid        `CEA` 180 kD GP                                                                        CEA 11-H5                                                                                 Wagener et al., 1984                      __________________________________________________________________________     abbreviations: Abs, antibodies; Ags, antigens; EGF, epidermal growth          factor; GI, gastrointestinal; GICA, gastrointestinalassociated antigen;       GP, glycoprotein; GY, gynecological; HMFG, human milk fat globule; Kd,        kilodaltons; Mabs, monoclonal antibodies; M.sub.r, molecular weight.; NS,     not specified; PLAP, placental alkaline phosphatase; TAG, tumorassociated     glycoprotein; CEA, carcinoembryonic antigen.                                  footnotes: the CA 199 Ag (GICA) is sialosylfucosyllactotetraosylceramide,     also termed sialylated Lewis pentaglycosyl ceramide or sialyated              lactoN-fucopentaose II; p97 Ags are believed to be chondroitin sulphate       proteoglycan; antigens reactive with Mab 9.2.27 are believed to be            sialylated glycoproteins associated with chondroitin sulphate                 proteoglycan; unless specified, GY can include cancers of the cervix,         #endocervix, endometrium, fallopian tube, ovary, vagina or mixed Mulleria     tumor; unless specified GI can include cancers of the liver, small            intestine, spleen, pancreas, stomach and oesophagus.                     

                  TABLE II                                                        ______________________________________                                        HUMAN TUMOR CELL LINES AND SOURCES                                            ATTC HTB                                                                      NUMBER   CELL LINE     TUMOR TYPE                                             ______________________________________                                        1       J82          Transitional-cell carcinoma, bladder                     2           RT4          Transitional-cell papilloma, bladder                 3           ScaBER    Squamous carcinoma, bladder                             4           T24          Transitional-cell carcinoma, bladder                 5           TCCSUP    Transitional-cell carcinoma, bladder,                                         primary grade IV                                        9           5637        Carcinoma, bladder, primary                           10         SK-N-MC   Neuroblastoma, metastasis to supra-                                            orbital area                                            11         SK-N-SH   Neuroblastoma, metastasis to bone                                               marrow                                                 12         SW 1088   Astrocytoma                                              13         SW 1783   Astrocytoma                                              14         U-87 MG   Glioblastoma, astrocytoma, grade III                     15         U-118 MG        Glioblastoma                                       16         U-138 MG        Glioblastoma                                       17         U-373 MG        Glioblastoma, astrocytoma, grade III               18         Y79           Retinoblastoma                                       19         BT-20       Carcinoma, breast                                      20         BT-474     Ductal carcinoma, breast                                22         MCF7         Breast adenocarcinoma, pleural                                               effusion                                               23         MDA-MB-134-VI                                                                            Breast, ductal carcinoma, pleural                                             effusion                                                24         MDA-MD-157                                                                                  Breast medulla, carcinoma, pleural                                         effusion                                                25         MDA-MB-175-VII                                                                          Breast, ductal carcinoma, pleural                                              effusion                                                27         MDA-MB-361                                                                                  Adenocarcinoma, breast, metastasis                                         to brain                                                30         SK-BR-3   Adenocarcinoma, breast, malignant                                               pleural effusion                                       31        C-33 A      Carcinoma, cervix                                       32         HT-3         Carcinoma, cervix, metastasis to                                            lymph node                                              33         ME-180     Epidermoid carcinoma, cervix,                                                 metastasis to omentum                                   34         MS751       Epidermoid carcinoma, cervix,                                                metastasis to lymph node                                35         SiHa         Squamous carcinoma, cervix                            36         JEG-3       Choriocarcinoma                                        37        Caco-2       Adenocarcinoma, colon                                  38        HT-29         Adenocarcinoma, colon, moderately                                            well-differentiated grade II                           39        SK-CO-1     Adenocarcinoma colon, ascites                           40        HuTu 80     Adenocarcinoma, duodenum                                41        A-253         Epidermoid carcinoma, submaxillary                                          gland                                                   43        FaDu           Squamous cell carcinoma, pharynx                     44        A-498         Carcinoma, kidney                                     45        A-704         Adenocarcinoma, kidney                                46        Caki-1       Clear cell carcinoma, consistent with                                        renal primary, metastasis to skin                       47        Caki-2       Clear cell carcinoma, consistent with                                        renal primary                                           48        SK-NEP-1   Wilms' tumor, pleural effusion                           49        SW 839       Adenocarcinoma, kidney                                 52        SK-HEP-1   Adenocarcinoma, liver, ascites                           53        A-427         Carcinoma, lung                                       54        Calu-1       Epidermoid carcinoma grade III,                                              lung, metastasis to pleura                              55        Calu-3       Adenocarcinoma, lung, pleural                                                effusion                                                56        Calu-6       Anaplastic carcinoma, probably lung                    57        SK-LU-1     Adenocarcinoma, lung consistent                                               with poorly differentiated, grade III                   58        SK-MES-1   Squamous carcinoma, lung, pleural                                              effusion                                                59        SW 900       Squamous cell carcinoma, lung                          60        EB1             Burkitt lymphoma, upper maxilla                     61        EB2             Burkitt lymphoma, ovary                             62        P3HR-1       Burkitt lymphoma, ascites                              63        HT-144       Malignant melanoma, metastasis to                                            subcutaneous tissue                                     64        Malme-3M         Malignant melanoma, metastasis to                                        lung                                                    66        RPMI-7951        Malignant melanoma, metastasis to                                        lymph node                                              67        SK-MEL-1   Malignant melanoma, metastasis to                                              lymphatic  system                                       68        SK-MEL-2   Malignant melanoma, metastasis to                                              skin of thigh                                           69        SK-MEL-3   Malignant melanoma, metastasis to                                              lymph node                                              70        SK-MEL-5         Malignant melanoma, metastasis to                                        axillary node                                           71        SK-MEL-24       Malignant melanoma, metastasis to                                         node                                                    72        SK-MEL-28       Malignant melanoma                                  73        SK-MEL-31       Malignant melanoma                                  75        Caov-3      Adenocarcinoma, ovary, consistent                                             with primary                                            76        Caov-4      Adenocarcinoma, ovary, metastasis                                             to subserosa of fallopian tube                          77        SK-OV-3    Adenocarcinoma, ovary, malignant                                               ascites                                                 78        SW 626      Adenocarcinoma, ovary                                   79        Capan-1    Adenocarcinoma, pancreas,                                                      metastasis to liver                                     80        Capan-2    Adenocarcinoma, pancrease                                81        DU 145      Carcinoma, prostate, metastasis to                                            brain                                                   82        A-204        Rhabdomyosarcoma                                       85        Saos-2      Osteogenic sarcoma, primary                             86        SK-ES-1    Anaplastic osteosarcoma versus                                                 Ewing sarcoma, bone                                     88        SK-LMS-1    Leiomyosarcoma, vulva, primary                          91        SW 684      Fibrosarcoma                                            92        SW 872      Liposarcoma                                             93        SW 982      Axilla synovial sarcoma                                 94        SW 1353    Chondrosarcoma, humerus                                  96        U-2 OS      Osteogenic sarcoma, bone primary                        102      Malme-3           Skin fibroblast                                    103      KATO III          Gastric carcinoma                                  104      Cate-1B     Embryonal carcinoma, testis,                                                   metastasis to lymph node                                105      Tera-1       Embryonal carcinoma, malignancy                                               consistent with metastasis to lung                      106      Tera-2       Embryonal carcinoma, malignancy                                               consistent with, metastasis to lung                     107      SW579         Thyroid carcinoma                                      111      AN3 CA       Endometrial adenocarcinoma,                                                   metastatic                                              112      HEC-1-A     Endometrial adenocarcinoma                               113      HEC-1-B     Endometrial adenocarcinoma                               114      SK-UT-1     Uterine, mixed mesodermal tumor,                                               consistent with leiomyosarcoma                                                grade III                                               115      SK-UT-1B    Uterine, mixed mesodermal tumor,                                               consistent with leiomyosarcoma                                                grade III                                               117      SW 954        Squamous cell carcinoma, vulva                         118      SW 962        Carcinoma, vulva, lymph node                                                 metastasis                                              119      NCI-H69      Small cell carcinoma, lung                              120      NCI-H128    Small cell carcinoma, lung                               121      BT-483        Ductal carcinoma, breast                               122      BT-549        Ductal carcinoma, breast                               123      DU4475        Metastatic cutaneous nodule, breast                                          carcinoma                                               124      HBL-100      Breast                                                  125      Hs 578Bst         Breast, normal                                     126      Hs 578T      Ductal carcinoma, breast                                127      MDA-MB-330       Carcinoma, breast                                   128      MDA-MB-415       Adenocarcinoma, breast                              129      MDA-MB-435S     Ductal carcinoma, breast                             130      MDA-MB-436       Adenocarcinoma, breast                              131      MDA-MB-453       Carcinoma, breast                                   132      MDA-MB-468       Adenocarcinoma, breast                              133      T-47D          Ductal carcinoma, breast, pleural                                           effusion                                                134      Hs 766T      Carcinoma, pancreas, metastatic to                                            lymph node                                              135      Hs 746T      Carcinoma, stomach, metastatic to                                             left leg                                                137      Hs 695T      Amelanotic melanoma, metastatic to                                            lymph node                                              138      Hs 683        Glioma                                                 140      Hs 294T      Melanoma, metastatic to lymph node                      142      Hs 602        Lymphoma, cervical                                     144      JAR              Choriocarcinoma, placenta                           146      Hs 445        Lymphoid, Hodgkin's disease                            147      Hs 700T      Adenocarcinoma, metastatic to pelvis                    148      H4                Neuroglioma, brain                                 151      Hs 696        Adenocarcinoma primary, unknown,                                             metastatic to bone-sacrum                               152      Hs 913T      Fibrosarcoma, metastatic to lung                        153      Hs 729        Rhabdomyosarcoma, left leg                             157      FHs 738Lu         Lung, normal fetus                                 158      FHs 173We         Whole embryo, normal                               160      FHs 738B1   Bladder, normal fetus                                    161      NIH:0VCAR-3      Ovary, adenocarcinoma                               163      Hs 67           Thymus, normal                                       166      RD-ES           Ewing's sarcoma                                      168      ChaGo K-1   Bronchogenic carcinoma                                                         subcutaneous metastasis, human                          169      WERI-Rb-1   Retinoblastoma                                           171      NCI-H446     Small cell carcinoma, lung                              172      NCI-H209     Small cell carcinoma, lung                              173      NCI-H146     Small cell carcinoma, lung                              174      NCI-H441     Papillary adenocarcinoma, lung                          175      NCI-H82       Small cell carcinoma, lung                             176      H9          T-cell lymphoma                                          177      NCI-H460     Large cell carcinoma, lung                              178      NCI-H596    Adenosquamous carcinoma, lung                            179      NCI-H676B   Adenocarcinoma, lung                                     180      NCI-H345     Small cell carcinoma, lung                              181      NCI-H820     Papillary adenocarcinoma, lung                          182      NCI-H520     Squamous cell carcinoma, lung                           183      NCI-H661     Large cell carcinoma, lung                              184      NCI-H510A   Small cell carcinoma,                                                          extra-pulmonary origin, metastatic                      185      D283 Med     Medulloblastoma                                         186      Daoy             Medulloblastoma                                     187      D341 Med     Medulloblastoma                                         188      AML-193       Acute monocyte leukemia                                189      MV4-11         Leukemia biphenotype                                  ______________________________________                                    

(a) Anti-Tumor Cell Antibodies

A straightforward means of recognizing a tumor antigen target is throughthe use of an antibody that has binding affinity for the particularantigen. An extensive number of antibodies are known that are directedagainst solid tumor antigens. Certain useful anti-tumor antibodies arelisted above in Table I. However, as will be instantly known to those ofskill in the art, certain of the antibodies listed in Table I will nothave the appropriate biochemical properties, or may not be of sufficienttumor specificity, to be of use therapeutically. An example is MUC8-22that recognizes a cytoplasmic antigen. Antibodies such as these willgenerally be of use only in investigational embodiments, such as inmodel systems or screening assays.

Generally speaking, antibodies for use in these aspects of the presentinvention will preferably recognize antigens that are accessible on thecell-surface and that are preferentially, or specifically, expressed bytumor cells. Such antibodies will also preferably exhibit properties ofhigh affinity, such as exhibiting a K_(d) of <200 nM, and preferably, of<100 nM, and will not show significant reactivity with life-sustainingnormal tissues, such as one or more tissues selected from heart, kidney,brain, liver, bone marrow, colon, breast, prostate, thyroid, gallbladder, lung, adrenals, muscle, nerve fibers, pancreas, skin, or otherlife-sustaining organ or tissue in the human body. The "life-sustaining"tissues that are the most important for the purposes of the presentinvention, from the standpoint of low reactivity, include heart, kidney,central and peripheral nervous system tissues and liver. The term"significant reactivity", as used herein, refers to an antibody orantibody fragment, that, when applied to the particular tissue underconditions suitable for immunohistochemistry, will elicit either nostaining or negligible staining with only a few positive cells scatteredamong a field of mostly negative cells.

Particularly promising antibodies from Table I contemplated for use inthe present invention are those having high selectivity for the solidtumor. For example, antibodies binding to TAG 72 and the HER-2proto-oncogene protein, which are selectively found on the surfaces ofmany breast, lung and colorectal cancers (Thor et al., 1986; Colcher etal., 1987; Shepard et al., 1991); MOv18 and OV-TL3 and antibodies thatbind to the milk mucin core protein and human milk fat globule (Miottiet al., 1985; Burchell et al., 1983); and the antibody 9.2.27 that bindsto the high M_(r) melanoma antigens (Reisfeld et al., 1982). Furtheruseful antibodies are those against the folate-binding protein, which isknown to be homogeneously expressed in almost all ovarian carcinomas;those against the erb family of oncogenes that are over-expressed insquamous cell carcinomas and the majority of gliomas; and otherantibodies known to be the subject of ongoing pre-clinical and clinicalevaluation.

The antibodies B3, KSI/4, CC49, 260F9, XMMCO-791, D612 and SM3 arebelieved to be particularly suitable for use in clinical embodiments,following the standard pre-clinical testing routinely practiced in theart. B3 (U.S. Pat. No. 5,242,813; Brinkmann et al., 1991) has ATCCAccession No. HB 10573; KS1/4 can be made as described in U.S. Pat. No.4,975,369; and D612 (U.S. Pat. No. 5,183,756) has ATCC Accession No. HB9796.

Another means of defining a tumor-associated target is in terms of thecharacteristics of the tumor cell, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,the inventors contemplate that any antibody that preferentially binds toa tumor cell listed in Table II may be used as the targeting componentof a bispecific ligand. The preferential tumor cell binding is againbased upon the antibody exhibiting high affinity for the tumor cell andnot having significant reactivity with life-sustaining normal cells ortissues, as defined above.

The invention therefore provides several means for generating anantibody for use in the targeted coagulation methods described herein.To generate a tumor cell-specific antibody, one would immunize an animalwith a composition comprising a tumor cell antigen and, as describedmore fully herein below, select a resultant antibody with appropriatespecificity. The immunizing composition may contain a purified, orpartially purified, preparation of any of the antigens in Table I; acomposition, such as a membrane preparation, enriched for any of theantigens in Table I; any of the cells listed in Table II; or a mixtureor population of cells that include any of the cell types listed inTable II.

Of course, regardless of the source of the antibody, in the practice ofthe invention in human treatment, one will prefer to ensure in advancethat the clinically-targeted tumor expresses the antigen ultimatelyselected. This is achieved by means of a fairly straightforward assay,involving antigenically testing a tumor tissue sample, for example, asurgical biopsy, or perhaps testing for circulating shed antigen. Thiscan readily be carried out in an immunological screening assay such asan ELISA (enzyme-linked immunosorbent assay), wherein the bindingaffinity of antibodies from a "bank" of hybridomas are tested forreactivity against the tumor. Antibodies demonstrating appropriate tumorselectivity and affinity are then selected for the preparation ofbispecific antibodies of the present invention.

Due to the well-known phenomenon of cross-reactivity, it is contemplatedthat useful antibodies may result from immunization protocols in whichthe antigens originally employed were derived from an animal, such as amouse or a primate, in addition to those in which the original antigenswere obtained from a human cell. Where antigens of human origin areused, they may be obtained from a human tumor cell line, or may beprepared by obtaining a biological sample from a particular patient inquestion. Indeed, methods for the development of antibodies that are"custom-tailored" to the patient's tumor are known (Stevenson et al.,1990) and are contemplated for use in connection with this invention.

(b) Further Tumor Cell Targets and Binding Ligands

In addition to the use of antibodies, other ligands could be employed todirect a coagulating agent to a tumor site by binding to a tumor cellantigen. For tumor antigens that are over-expressed receptors (oestrogenreceptor, EGF receptor), or mutant receptors, the corresponding ligandscould be used as targeting agents.

In an analogous manner to endothelial cell receptor ligands, there maybe components that are specifically, or preferentially, bound to tumorcells. For example, if a tumor antigen is an over-expressed receptor,the tumor cell may be coated with a specific ligand in vivo. It seemsthat the ligand could then be targeted either with an antibody againstthe ligand, or with a form of the receptor itself. Specific examples ofthese type of targeting agents are antibodies against TIE-1 or TIE-2ligands, antibodies against platelet factor 4, and leukocyte adhesionbinding protein.

2. Other Disease Targets

In further embodiments, the first binding region may be a component thatbinds to a target molecule that is specifically or preferentiallyexpressed in a disease site other than a tumor site.

Exemplary target molecules associated with other diseased cells include,for example, leukocyte adhesion molecules, that are associated withpsoriasis; FGF, that is associated with proliferative diabeticretinopathy; platelet factor 4, that is associated with the activatedendothelium of various diseases; and VEGF, that is associated withvascular proliferative disease. It is believed that an animal or patienthaving any one of the above diseases would benefit from the specificinduction of coagulation in the disease site.

Diseases that are known to have a common angio-dependent pathology, asdescribed in Klagsburn & Folkman (1990), may also be treated withbispecific ligand as described herein. In particular, a vascularendothelial cell-targeted ligand or a stroma-targeted ligand will beused to achieve coagulation in the disease site. The treatment of BPH,diabetic retinopathy, vascular restenosis, vascular adhesions, AVM,meningioma, hemangioma, neovascular glaucoma, rheumatoid arthritis andpsoriasis are particularly contemplated at the present time.

3. Disease-Associated Vasculature Cell Targets

The cells of the vasculature are intended as targets for use in thepresent invention. In these cases, one binding region of the bispecificligand will be capable of binding to an accessible marker preferentiallyexpressed by disease-associated vasculature endothelial cells. Theexploitation of the vascular markers is made possible due to theproximity of the vascular endothelial cells to the disease area and tothe products of the local aberrant physiological processes. For example,tumor vascular endothelial cells are exposed to tumor cells andtumor-derived products that change the phenotypic profile of theendothelial cells.

Tumor cells are known to elaborate tumor-derived products, such aslymphokines, monokines, colony-stimulating factors, growth factors andangiogenic factors, that act on the nearby vascular endothelial cells(Kandel et al., 1991; Folkman, 1985a,b) and cytokines (Burrows et al.,1991; Ruco et al., 1990; Borden et al., 1990). The tumor products bindto the endothelial cells and serve to selectively induce expression ofcertain molecules. It is these induced molecules that may be targetedusing the tumor endothelium-specific coagulant delivery provided bycertain aspects of the present invention. Vascular endothelial cells intumors proliferate at a rate 30-fold greater than those in miscellaneousnormal tissues (Denekamp et al., 1982), suggesting thatproliferation-linked determinants could also serve as markers for tumorvascular endothelial cells.

In certain embodiments of the invention, the targeting component of thebispecific ligands will be a component that has a relatively high degreeof specificity for tumor vasculature. These targeting components may bedefined as components that bind to molecules expressed on tumorendothelium, but that have little or no expression at the surface ofnormal endothelial cells. Such specificity may be assessed by thestandard procedures of immunostaining of tissue sections, which areroutine to those of skill in the art.

However, as stated above, an advantage of the present invention is thatthe requirement for selectivity is not as stringent as previously neededin the prior art methods, especially those employing immunotoxins,because any side effects associated with the mis-targeting of thecoagulating agent will be minimal in comparison to those resulting fromthe mis-targeting of a toxin.

Therefore, it is generally proposed that the molecules to be targetedusing the bispecific ligands or antibodies of this invention will bethose that are expressed on tumor vasculature at a higher level than onnormal endothelial cells.

(a) Vascular Endothelial Cell Markers in Disease

Molecules that are known to be preferentially expressed at the surfaceof vascular endothelial cells in a disease site or environment areherein termed "natural disease-associated vascular endothelial cellmarkers". This term is used for simplicity to refer to the endothelialcell components that are expressed in diseases connected with increasedor inappropriate angiogenesis or endothelial cell proliferation. Oneparticular example are the tumor endothelial cell components that areexpressed in situ in response to tumor-derived factors. These componentsare also termed "naturally-induced tumor endothelial cell markers".

Both VEGF/VPF (vascular endothelial growth factor/vascular permeabilityfactor) and components of the FGF (fibroblast growth factor) family areconcentrated in or on tumor vasculature. The corresponding receptorstherefore provide a potential target for attack on tumor vasculature.For example, VEGF receptors are known to be upregulated on tumorendothelial cells, as opposed to endothelial cells in normal tissues,both in rodents and man (Thieme et al., 1995). Possibly, this is aconsequence of hypoxia--a characteristic of the tumor microenvironment(Leith et al., 1992). FGF receptors are also upregulated three-fold onendothelial cells exposed to hypoxia, and so are believed to beupregulated in tumors (Bicknell and Harris et al., 1992).

The TGF β (transforming growth factor β) receptor (endoglin) onendothelial cells is upregulated on dividing cells, providing anothertarget. One of the present inventors found that endoglin is upregulatedon activated and dividing HUVEC in culture, and is strongly expressed inhuman tissues on endothelial cells at sites of neovascularization,including a broad range of solid tumors and fetal placenta. In contrast,endothelial cells in the majority of miscellaneous non-malignant adulttissues, including preneoplastic lesions, contain little or no endoglin.Importantly, endoglin expression is believed to correlate withneoplastic progression in the breast, as shown by benign fibroadenomasand early carcinomas binding low levels of TEC-4 and TEC-11 antibodies,and late stage intraductal carcinomas and invasive carcinomas bindinghigh levels of these antibodies.

Other natural disease-associated vascular endothelial cell markersinclude a TIE, VCAM-1, P-selectin, E-selectin, α_(v) β₃ integrin,pleiotropin and endosialin, each of which may be targeted using theinvention.

(b) Cytokine-Inducible Vascular Endothelial Markers

Due to the nature of disease processes, which often result in localizeddysfunction within the body, methods are available to manipulate thedisease site whilst leaving other tissues relatively unaffected. This isparticularly true in malignant and benign tumors, which exist asdistinct entities within the body of an animal. For example, the tumorenvironment may be manipulated to create additional markers that arespecific for tumor vascular endothelial cells. These methods generallymimic those that occur naturally in solid tumors, and also involve thelocal production of signalling agents, such as growth factors orcytokines, that induce the specific expression of certain molecules atthe surface of the nearby vascular endothelial cells.

The group of molecules that may be artificially induced to be expressedat the surface of vascular endothelial cells in a disease or tumorenvironment are herein termed "inducible endothelial cell markers", orspecifically, inducible tumor endothelial cell markers. This term isused to refer to those markers that are artificially induced, i.e.,induced as a result of manipulation by the hand of man, rather thanthose that are induced as part of the disease or tumor developmentprocess in an animal. The term "inducible marker", as defined above, ischosen for simple reference in the context of the present application,notwithstanding the fact that "natural markers" are also induced, e.g.,by tumor-derived agents.

Thus, although not required to practice the invention, techniques forthe selective elicitation of vascular endothelial antigen targets on thesurface of disease-associated vasculature are available that may, ifdesired, be used in conjunction with the invention. These techniquesinvolve manipulating the antigenic expression, or cell surfacepresentation, such that a target antigen is expressed or renderedavailable on the surface of disease-associated vasculature and notexpressed or otherwise rendered accessible or available for binding, orat least to a lesser extent, on the surface of normal endothelium.

Tumor endothelial markers can be induced by tumor-derived cytokines(Burrows et al., 1991; Ruco et al., 1990) and by angiogenic factors(Mignatti et al., 1991). Examples of cell surface markers that may bespecifically induced in the tumor endothelium and then targeted using abispecific coagulating ligand, as provided by the invention, includethose listed in Table III (Bevilacqua et al., 1987; Dustin et al., 1986;Osborn et al., 1989; Collins et al., 1984).

The mechanisms for the induction of the proposed markers; the inducing,or "intermediate cytokine", such as IL-1 and IFN-γ; and the leukocytecell type and associated cytokine-activating molecule, whose targetingwill result in the release of the cytokine, are also set forth in TableIII. In the induction of a specific marker, a bispecific"cytokine-inducing" or "antigen-inducing" antibody is generallyrequired. This antibody will selectively induce the release of theappropriate cytokine in the locale of the tumor, thus selectivelyinducing the expression of the desired target antigen by the vascularendothelial cells. The bispecific antibody cross-links cells of thetumor mass and cytokine-producing leukocytes, thereby activating theleukocytes to release the cytokine.

The preparation and use of bispecific antibodies such as these ispredicated in part on the fact that cross-linking antibodies recognizingCD3, CD14, CD16 and CD28 have previously been shown to elicit cytokineproduction selectively upon cross-linking with the second antigen (Qianet al., 1991). In the context of the present invention, since onlysuccessfully tumor cell-crosslinked leukocytes will be activated torelease the cytokine, cytokine release will be restricted to the localeof the tumor. Thus, expression of the desired marker, such asE-selectin, will be similarly limited to the endothelium of the tumorvasculature.

                                      TABLE III                                   __________________________________________________________________________    POSSIBLE INDUCIBLE VASCULAR TARGETS                                                                            LEUKOCYTES                                                                            LEUKOCYTE MOLECULES WHICH,                                                   WHICH                                                                              WHEN CROSSLINKED BY              INDUCIBLE            SUBTYPES/ALIASES                                                                                 PRODUCE                                                                          MONOCLONAL ANTIBODIES              ENDOTHELIAL                                                                                            (MOLECULAR                                                                          INDUCING                                                                          THOSE    ACTIVATE THE CELLS TO             CELL MOLECULES                                                                          ACRONYM                                                                               FAMILY)         CYTOKINESES                                                                           PRODUCE CYTOKINES                   __________________________________________________________________________    Endothelial-                                                                            ELAM-1                                                                              --        IL-1, TNF-                                                                           monocytes                                                                             CD14                                 Leukocyte             (Selectin)                                                                         α, (TNF-β)                                                               macrophages                                                                            CD14                                Adhesion             lect-                                                                                      mast cellsrial                                                                         FcR for IgE                        Molecule-1                                                                                                          Endotoxin)                              Vascular Cell                                                                               VCAM-1                                                                            Inducible Cell                                                                            (Bacterial                                                                        monocytes                                                                                  CD14                           Adhesion                Adhesion                                                                               macrophagesn)                                                                             CD14                             Molecule-1                                                                                                    IL-1, TNF-                                                                      mast cells                                                                               FcR for IgE                                                       α)                                                                 TNF-β, IL-4n                                                                  helper T cells                                                                          CD2, CD3, CD28                                                           NK cells                                                                                    FcR for IgG (CD16)        Intercellular                                                                                ICAM-1                                                                               --         monocytes1, TNFα                                                                     CD14                            Adhesion              (Immunoglobulin                                                                        (Bacterial                                                                         macrophages                                                                             CD15                            Molecule-1                                                                                          Family                                                                                      mast cellsxin)                                                                          FcR for IgE                                                          T helper cellsa.,                                                                  CD2, CD3, CD28                                                              NK cellsamma.                                                                        FcR for IgG (CD16)             The Agent for                                                                                LAM-1                                                                            MEL-14 Agent                                                                                 monocytes-1, TNFα                                                                     CD14                           Leukocyte          (Mouse)                                                                                          macrophages                                                                         CD14                              Adhesion                             mast cellstoxin)                                                                      FcR for IgE                      Molecule-1                                                                    Major                  HLA-DR                                                                                       helper T cells                                                                     CD2, CD3, CD28                     Histocompatabi-                                                                            Class                                                                                HLA-DP-Human                                              lity Complex                                                                                  II                                                                                   HLA-DQ                                                 Clasds II                 I-A-Mouse                                                                                            FcR for IgG (CD16)           Antigen                   I-E                                                 __________________________________________________________________________

It is important to note that, from the possible inducible markers listedin Table III, E-selectin and MHC Class II antigens, such as HLA-DR,HLA-DP and HLA-DQ (Collins et al., 1984), are by far the most preferredtargets for use in connection with clinical embodiments. The otheradhesion molecules of Table III appear to be expressed to varyingdegrees in normal tissues, generally in lymphoid organs and onendothelium, making their targeting perhaps appropriate only in animalmodels or in cases where their expression on normal tissues can beinhibited without significant side-effects. The targeting of E-selectinor an MHC Class II antigen is preferred as the expression of theseantigens will likely be the most direct to promote selectively intumor-associated endothelium.

E-selectin

The targeting of an antigen that is not expressed on the surfaces ofnormal endothelium is the most straightforward form of the inductionmethods. E-selectin is an adhesion molecule that is not expressed innormal endothelial vasculature or other human cell types (Cotran et al.,1986), but can be induced on the surface of endothelial cells throughthe action of cytokines such as IL-1, TNF, lymphotoxin and bacterialendotoxin (Bevilacqua et al., 1987). It is not induced by IFN-γ (Wu etal., 1990). The expression of E-selectin may thus be selectively inducedin tumor endothelium through the selective delivery of such a cytokine,or via the use of a composition that causes the selective release ofsuch cytokines in the tumor environment.

Bispecific antibodies are one example of a composition capable ofcausing the selective release of one or more of the foregoing or otherappropriate cytokines in the tumor site, but not elsewhere in the body.Such bispecific antibodies are herein termed "antigen-inducingantibodies" and are, of course, distinct from any bispecific antibodiesof the invention that have targeting and coagulating components.Antigen-inducing antibodies are designed to cross-link cytokine effectorcells, such as cells of monocyte/macrophage lineage, T cells and/or NKcells or mast cells, with tumor cells of the targeted solid tumor mass.This cross-linking would then effect a release of cytokine that islocalized to the site of cross-linking, i.e., the tumor.

Effective antigen-inducing antibodies recognize a selected tumor cellsurface antigen on the one hand (e.g., those in Table I) and, on theother hand, recognize a selected "cytokine activating" antigen on thesurface of a selected leukocyte cell type. The term "cytokineactivating" antigen is used to refer to any one of the various knownmolecules on the surfaces of leukocytes that, when bound by an effectormolecule, such as an antibody or a fragment thereof or anaturally-occurring agent or synthetic analog thereof, be it a solublefactor or membrane-bound counter-receptor on another cell, promotes therelease of a cytokine by the leukocyte cell. Examples of cytokineactivating molecules include CD14 (the LPS receptor) and FcR for IgE,which will activate the release of IL-1 and TNFα; and CD16, CD2 or CD3or CD28, which will activate the release of IFNγ and TNFβ, respectively.

Once introduced into the bloodstream of an animal bearing a tumor, suchan antigen-inducing bispecific antibody will bind to tumor cells withinthe tumor, cross-link those tumor cells with effector cells, e.g.,monocytes/macrophages, that have infiltrated the tumor, and thereaftereffect the selective release of cytokine within the tumor. Importantly,however, without cross-linking of the tumor and leukocyte, theantigen-inducing antibody will not effect the release of cytokine. Thus,no cytokine release will occur in parts of the body removed from thetumor and, hence, expression of cytokine-induced molecules, e.g.,E-selectin, will occur only within the tumor endothelium.

A number of useful "cytokine activating" antigens are known, which, whencross-linked with an appropriate bispecific antibody, will result in therelease of cytokines by the cross-linked leukocyte. The generallypreferred target for this purpose is CD14, which is found on the surfaceof monocytes and macrophages. When CD14 is cross linked it stimulatesmonocytes/macrophages to release IL-1 (Schutt et al., 1988; Chen et al.,1990), and possibly other cytokines, which, in turn stimulate theappearance of E-selectin on nearby vasculature. Other possible targetsfor cross-linking in connection with E-selectin induction and targetinginclude FcR for IgE, found on Mast cells; FcR for IgG (CD16), found onNK cells; as well as CD2, CD3 or CD28, found on the surfaces of T cells.Of these, CD14 targeting is generally preferred due to the relativeprevalence of monocyte/macrophage infiltration of solid tumors asopposed to the other leukocyte cell types.

In an exemplary induction embodiment, an animal bearing a solid tumor isinjected with bispecific (Fab'--Fab') anti-CD14/anti-tumor antibody(such as anti-CEA, 9.2.27 antibody against high Mr melanoma antigensOV-TL3 or MOv 18 antibodies against ovarian associated antigens). Theantibody localizes in the tumor, by virtue of its tumor bindingactivity, and then activates monocytes and macrophages in the tumor bycrosslinking their CD14 antigens (Schutt et. al., 1988; Chen et. al.,1990). The activated monocytes/macrophages have tumoricidal activity(Palleroni et. al., 1991) and release IL-1 and TNF which rapidly induceE-selectin antigens on the tumor vascular endothelial cells (Bevilacquaet. al., 1987; Pober et. al., 1991).

MHC Class II Antigens

The second preferred group of inducible markers contemplated for usewith the present invention are the MHC Class II antigens (Collins etal., 1984), including HLA-DR, HLA-DP and HLA-DQ. Class II antigens areexpressed on vascular endothelial cells in most normal tissues inseveral species, including man. Studies in vitro (Collins et al., 1984;Daar et al., 1984; O'Connell et al., 1990) and in vivo (Groenewegen etal., 1985) have shown that the expression of Class II antigens byvascular endothelial cells requires the continuous presence of IFN-γwhich is elaborated by T_(H1) cells and, to a lesser extent, by NK cellsand CD8⁺ T cells.

MHC Class II antigens are not unique to vascular endothelial cells, andare also expressed constitutively on B cells, activated T cells, cellsof monocyte/macrophage linage and on certain epithelial cells, both inmice (Hammerling, 1976) and in man (Daar et al., 1984). Due to theexpression of MHC Class II antigens on "normal" endothelium, theirtargeting is not quite so straightforward as E-selectin. However, theinduction and targeting of MHC Class II antigens is made possible byusing in conjunction with an immunosuppressant, such as Cyclosporin A(CsA), that has the ability to effectively inhibit the expression ofClass II molecules in normal tissues (Groenewegen et al., 1985). The CsAacts by preventing the activation of T cells and NK cells (Groenewegenet al., 1985; DeFranco, 1991), thereby reducing the basal levels ofIFN-γ below those needed to maintain Class II expression on endothelium.

There are various other cyclosporins related to CsA, includingcyclosporins A, B, C, D, G, and the like, that also haveimmunosuppressive action and are likely to demonstrate an ability tosuppress Class II expression. Other agents that might be similarlyuseful include FK506 and rapamycin.

Thus, the practice of the MHC Class II induction and targetingembodiment requires a pretreatment of the tumor-bearing animal with adose of CsA or other Class II immunosuppressive agent that is effectiveto suppress Class II expression. In the case of CsA, this will typicallybe on the order of about 10 to about 30 mg/kg body weight. Oncesuppressed in normal tissues, Class II antigens can then be selectivelyinduced in the tumor endothelium, again through the use of a bispecificantibody.

In this case, the antigen-inducing bispecific antibody will havespecificity for a tumor cell marker and for an activating antigen foundon the surface of an effector cell that is capable of inducing IFN-γproduction. Such effector cells will generally be helper T cells (T_(H))or Natural Killer (NK) cells. In these embodiments, it is necessary thatT cells, or NK cells if CD16 is used, be present in the tumor to producethe cytokine intermediate in that Class II antigen expression isachieved using IFN-γ, but is not achieved with the other cytokines.Thus, for the practice of this aspect of the invention, one will desireto select CD2, CD3, CD28, or most preferably CD28, as the cytokineactivating antigen for targeting by the antigen-inducing bispecificantibody.

The T cells that should be activated in the tumor are those adjacent tothe vasculature since this is the region most accessible to cells and isalso where the bispecific antibody will be most concentrated. Theactivated T cells should then secrete IFN-γ which induces Class IIantigens on the adjacent tumor vasculature.

The use of a bispecific (Fab'--Fab') antibody having one arm directedagainst a tumor antigen and the other arm directed against CD28 iscurrently preferred. This antibody will crosslink CD28 antigens on Tcells in the tumor which, when combined with a second signal (provided,for example, by IL-1 which is commonly secreted by tumor cells (Burrowset al., 1991; Ruco et al., 1990), has been shown to activate T cellsthrough a CA²⁺ -independent non-CsA-inhibitable pathway (Hess et al.,1991; June et al., 1987; Bjorndahl et al., 1989).

The preparation of antibodies against various cytokine activatingmolecules is also well known in the art. For example, the preparationand use of anti-CD14 and anti-CD28 monoclonal antibodies having theability to induce cytokine production by leukocytes has now beendescribed by several laboratories (reviewed in Schutt et al., 1988; Chenet al., 1990, and June et al., 1990, respectively). Moreover, thepreparation of monoclonal antibodies that will stimulate leukocyterelease of cytokines through other mechanisms and other activatingantigens is also known (Clark et al., 1986; Geppert et al., 1990).

In still further embodiments, the inventors contemplate an alternativeapproach for suppressing the expression of Class II molecules, andselectively eliciting Class II molecule expression in the locale of thetumor. This approach, which avoids the use of both CsA and a bispecificactivating antibody, takes advantage of the fact that the expression ofClass II molecules can be effectively inhibited by suppressing IFN-γproduction by T cells, e.g., through use of an anti-CD4 antibody (Streetet al., 1989). Using this embodiment, IFN-γ production is inhibited byadministering anti-CD4, resulting in the general suppression of Class IIexpression. Class II is then induced only in the tumor site, e.g., usingtumor-specific T cells which are only activatable within the tumor.

In this mode of treatment, one will generally pretreat an animal orhuman patient with a dose of anti-CD4 that is effective to suppressIFN-γ production and thereby suppress the expression of Class IImolecules. Effective doses are contemplated to be, for example, on theorder of about 4 to about 10 mg/kg body weight. After Class IIexpression is suppressed, one will then prepare and introduce into thebloodstream an IFN-γ-producing T cell clone (e.g., T_(h) 1 or cytotoxicT lymphocyte, CTL) specific for an antigen expressed on the surface ofthe tumor cells. These T cells localizes to the tumor mass, due to theirantigen recognition capability and, upon such recognition, then releaseIFN-γ. In this manner, cytokine release is again restricted to thetumor, thus limiting the expression of Class II molecules to the tumorvasculature.

The IFN-γ-producing T cell clone may be obtained from the peripheralblood (Mazzocchi et al., 1990), however, a preferred source is fromwithin the tumor mass (Fox et al., 1990). The currently preferred meansof preparing such a T cell clone is to remove a portion of the tumormass from a patient; isolate cells, using collagenase digestion, wherenecessary; enrich for tumor infiltrating leukocytes using densitygradient centrifugation, followed by depletion of other leukocytesubsets by, e.g., treatment with specific antibodies and complement; andthen expand the tumor infiltrating leukocytes in vitro to provide theIFN-γ producing clone. This clone will necessarily be immunologicallycompatible with the patient, and therefore should be well tolerated bythe patient.

It is proposed that particular benefits will be achieved by furtherselecting a high IFN-γ producing T cell clone from the expandedleukocytes by determining the cytokine secretion pattern of eachindividual clone every 14 days. To this end, rested clones will bemitogenically or antigenically-stimulated for about 24 hours and theirculture supernatants assayed, e.g., using a specific sandwich ELISAtechnique (Cherwinski et al., 1989), for the presence of IL-2, IFN-γ,IL-4, IL-5 and IL-10. Those clones secreting high levels of IL-2 andIFN-γ, the characteristic cytokine secretion pattern of T_(H1) clones,will be selected. Tumor specificity will be confirmed usingproliferation assays.

Furthermore, one will prefer to employ as the anti-CD4 antibody ananti-CD4 Fab, because it will be eliminated from the body within 24hours after injection and so will not cause suppression of thetumor-recognizing T-cell clones that are subsequently administered. Thepreparation of T cell clones having tumor specificity is generally knownin the art, as exemplified by the production and characterization of Tcell clones from lymphocytes infiltrating solid melanoma tumors (Maedaet al., 1991).

In using either of the MHC Class II suppression-induction methods,additional benefits will likely result from the fact that anti-Class IIantibodies injected intravenously do not appear to reach the epithelialcells or the monocytes/macrophages in normal organs other than the liverand spleen. Presumably this is because the vascular endothelium in mostnormal organs is tight, not fenestrated as it is in the liver andspleen, and so the antibodies must diffuse across basement membranes toreach the Class II-positive cells. Also, any B cell elimination that mayresult, e.g., following cross-linking, is unlikely to pose a significantproblem as these cells are replenished from Class II negativeprogenitors (Lowe et al., 1986). Even B cell killing, as occurs in Blymphoma patients, causes no obvious harm (Vitetta et al., 1991).

In summary, although the tumor coagulating compositions and antibodiesof the present invention are elegantly simple, and do not require theinduction of antigens for their operability, the combined use of anantigen-inducing bispecific antibody with this invention is alsocontemplated. Such antibodies would generally be administered prior tothe bispecific coagulating ligands of this invention.

Generally speaking, the more "immunogenic" tumors would be more suitablefor the MHC Class II approach involving, e.g., the cross-linking of Tcells in the tumor through an anti-CD28/anti-tumor bispecific antibody,because these tumors are more likely to be infiltrated by T cells, aprerequisite for this method to be effective. Examples of immunogenicsolid tumors include renal carcinomas, melanomas, a minority of breastand colon cancers, as well as possibly pancreatic, gastric, liver, lungand glial tumor cancers. These tumors are referred to as "immunogenic"because there is evidence that they elicit immune responses in the hostand they have been found to be amenable to cellular immunotherapy(Yamaue et al., 1990). In the case of melanomas and large bowel cancers,the most preferred antibodies for use in these instances would be B72.3(anti-TAG-72) and PRSC5/PR4C2 (anti-Lewis a) or 9.2.27 (anti-high Mrmelanoma antigen).

For the majority of solid tumors of all origins, an anti-CD14 approachthat employs a macrophage/monocyte intermediate would be more suitable.This is because most tumors are rich in macrophages. Examples ofmacrophage-rich tumors include most breast, colon and lung carcinomas.Examples of preferred anti-tumor antibodies for use in these instanceswould be anti-HER-2, B72.3, SM-3, HMFG-2, and SWA11 (Smith et al.,1989).

(c) Coagulant-Inducible Markers

Coagulants, such as thrombin, Factor IX/IXa, Factor X/Xa, plasmin andmetalloproteinases, such as interstitial collagenases, stromelysins andgelatinases, also act to induce certain markers. In particular,E-selectin, P-selectin, PDGF and ICAM-1 are induced by thrombin (Sugamaet. al., 1992; Shankar et. al., 1994).

Therefore, for this induction, an anti-coagulant/anti-tumor bispecificantibody will be utilized. The antibody will localize in the tumor viaits tumor binding activity. The bispecific will then concentrate thecoagulant, e.g., thrombin, in the tumor, resulting in induction ofE-selectin and P-selectin on the tumor vascular endothelial cells(Sugama et. al., 1991; Shankar et. al., 1994).

Alternatively, targeting of truncated tissue factor to tumor cells orendothelium will induce thrombin deposition within the tumor. As thethrombin is deposited, E-selectin and P-selectin will be induced on thetumor vascular endothelial cells.

(d) Antibodies to Vascular Endothelial Cell Markers

A straightforward means of recognizing a disease-associated vasculaturetarget, whether induced in the natural environment or by artificialmeans, is through the use of an antibody that has binding affinity forthe particular cell surface receptor, molecule or antigen. These includeantibodies directed against all cell surface components that are knownto be present on, e.g., tumor vascular endothelial cells, those that areinduced or over-expressed in response to tumor-derived factors, andthose that are induced following manipulation by the hand of man. TableIV and Table V summarize useful antibodies and their properties.

                                      TABLE IV                                    __________________________________________________________________________    SUMMARY OF VASCULATURE STAINING PATTERNS                                      OF CERTAIN ANTIBODIES TO HUMAN TUMOR VASCULATURE                                                  % Tumor types                                                                        % tumor vessels                                                                       normal vessel                              Antibody                                                                                           stainednce                                                                                 stained                                                                               reactivity                          __________________________________________________________________________    anti-vWF                                                                           VIII R Ag      100    100     strong on all                              FB5                       30&in                                                                                  10-20                                                                              lymphoid organs                                                        old                                          TP3              Brulandsteosarcoma                                                                      50                                                                                   10-30                                                                               strong on small                                                                           BV                                                  protein                                             BC-1           fibronectin isoform                                                              Zardi                                                                                          10-30                                                                                    none                            TV-1                       100                                                                                           strong on all                      LM 609                                                                              α.sub.v β.sub.e vitronectin                                                 Cheneoh                                                                                   85                                                                                           medium on all                                                 receptor                                            TEC 11                                                                                                                   weak on most                       TEC 110                                                                                                                  weak on most                       __________________________________________________________________________

                                      TABLE V                                     __________________________________________________________________________    COMPARISON OF ANTI-EC mAbs                                                    ON HUMAN TUMORS                                                               TUMOR TYPE                                                                             n TEC 110                                                                            TEC 11                                                                            FB-5                                                                             TP-3  BC-1                                                                             TV-1                                                                             LM 609                                     __________________________________________________________________________    DIGESTIVE                                                                     Gastrointestinal                                                                                                       ++                                   Parotid                                 ND                                    REPRODUCTIVE                                                                  Breast                                   -                                    Ovary                            ++MALL)                                                                               +                                    Uterus                                   ++                                   RESPIRATORY                                                                   Lung                                     +                                    LYMPHOID                                                                      Hodgkins                                +-                                    __________________________________________________________________________

Two further antibodies that may be used in this invention are thosedescribed by Rettig et al. (1992) and Wang et al. (1993) that aredirected against unrelated antigens of unknown function expressed in thevasculature of human tumors, but not in most normal tissues.

The antibody described by Kim et. al. (1993) may also be used in thisinvention, particularly as this antibody inhibited angiogenesis andsuppressed tumor growth in vivo.

Antibodies that have not previously been shown to be specific for humantumors may also be used. For example, Venkateswaran et al. (1992)described the production of anti-FGF MAbs. Xu et. al. (1992) developedand characterized a panel of 16 isoform and domain-specific polyclonaland monoclonal antibodies against FGF receptor (flg) isoforms. Massogliaet al. (1987) also reported MAbs against the against FGF.

(e) Generation of Antibodies to Disease Vasculature

In addition to utilizing a known antibody, such as those described aboveand others known and published in the scientific literature, one mayalso generate a novel antibody using standard immunization procedures,as described in more detail hereinbelow. To generate an antibody againsta known disease-associated vascular marker antigen, one would immunizean animal with an immunogenic composition comprising the antigen. Thismay be a membrane preparation that includes, or is enriched for, theantigen; a relatively purified form of the antigen, as isolated fromcells or membranes; a highly purified form of the antigen, as obtainedby a variety of purification steps using, e.g., a native antigen extractor a recombinant form of the antigen obtained from a recombinant hostcell.

The present invention also provides yet further methods for generatingan antibody against an antigen present on disease-associated vasculatureendothelial cells, which methods are suitable for use even where thebiochemical identity of the antigen remains unknown. These methods areexemplified through the generation of an antibody against tumorvasculature endothelial cells. A first means of achieving antibodygeneration in this manner uses a preparation of vascular endothelialcells obtained from the tumor site of an animal or human patient. Onesimply immunizes an experimental animal with a preparation of such cellsand collects the antibodies so produced. The most useful form of thismethod is that where specific antibodies are subsequently selected, asmay be achieved using conventional hybridoma technology and screeningagainst tumor vascular endothelial cells.

A development of the above method is that which mimics the tumorvasculature phenomenon in vitro, and where cell purification is notnecessary. In using this method, endothelial cells are subjected totumor-derived products, such as might be obtained from tumor-conditionedmedia, in cell culture rather than in an animal. This method generallyinvolves stimulating endothelial cells with tumor-conditioned medium andemploying the stimulated endothelial cells as immunogens to prepare acollection of antibodies. Again, specific antibodies should be selected,e.g., using conventional monoclonal antibody technology, or othertechniques such as combinatorial immunoglobulin phagemid librariesprepared from RNA isolated from the spleen of the immunized animal. Onewould select a specific antibody that preferentially recognizestumor-stimulated vascular endothelium and reacts more strongly withtumor-associated endothelial cells than with normal adult human tissues.

Stimulated endothelial cells contemplated to be of use in this regardinclude, for example, human umbilical vein endothelial cells (HUVE),human dermal microvascular endothelial cells (HDEMC), human saphenousvein endothelial cells, human omental fat endothelial cells, other humanmicrovascular endothelial cells, human brain capillary endothelialcells, and the like. It is also contemplated that endothelial cells fromanother species may stimulated by tumor-conditioned media and employedas immunogens to generate hybridomas to produce an antibodies inaccordance herewith, i.e., to produce antibodies that crossreact withtumor-stimulated human vascular endothelial cells, and/or antibodies foruse in pre-clinical models.

"Tumor-conditioned medium or media" are defined herein as compositionsor media, such as culture media, that contain one or more tumor-derivedcytokines, lymphokines or other effector molecules. Most typically,tumor-conditioned medium is prepared from a culture medium in whichselected tumor cells have been grown, and will therefore be enriched insuch tumor-derived products. The type of medium is not believed to beparticularly important, so long as it at least initially containsappropriate nutrients and conditions to support tumor cell growth. It isalso, of course, possible to extract and even separate materials fromtumor-conditioned media and employ one or more of the extracted productsfor application to the endothelial cells.

As for the type of tumor used for the preparation of the medium ormedia, one will, of course, prefer to employ tumors that mimic orresemble the tumor that will ultimately be subject to analysis ortreatment using the present invention. Thus, for example, where oneenvisions the development of a protocol for the treatment of breastcancer, one will desire to employ breast cancer cells such as ZR-75-1,T47D, SKBR3, MDA-MB-231. In the case of colorectal tumors, one maymention by way of example the HT29 carcinoma, as well as DLD-1, HCT116or even SW48 or SW122. In the case of lung tumors, one may mention byway of example NCI-H69, SW2, NCI H23, NCI H460, NCI H69, or NCI H82. Inthe case of melanoma, good examples are DX.3, A375, SKMEL-23, HMB-2,MJM, T8 or indeed VUP. In any of the above cases, it is further believedthat one may even employ cells produced from the tumor that is to betreated, i.e., cells obtained from a biopsy.

Once prepared, the tumor-conditioned media is then employed to stimulatethe appearance of tumor endothelium-specific marker(s) on the cellsurfaces of endothelial cells, e.g., by culturing selected endothelialcells in the presence of the tumor-conditioned media (or productsderived therefrom). Again, it is proposed that the type of endothelialcell that is employed is not of critical importance, so long as it isgenerally representative of the endothelium associated with thevasculature of the particular tumor that is ultimately to be treated ordiagnosed. The inventors prefer to employ human umbilical veinendothelial cells (HUVE), or human dermal microvascular endothelialcells (HDMEC, Karasek, 1989), in that these cells are of human origin,respond to cytokine growth factors and angiogenic factors and arereadily obtainable. However, it is proposed that any endothelial cellthat is capable of being cultured in vitro may be employed in thepractice of the invention and nevertheless achieve beneficial results.One may mention, by way of example, cells such as EA.hy9.26, ECV304,human saphenous vein endothelial cells, and the like.

Once stimulated using the tumor-derived products, the endothelial cellsare then employed as immunogens in the preparation of monoclonalantibodies (MAbs). The technique for preparing MAbs against antigeniccell surface markers is quite straightforward, and may be readilycarried out using techniques well known to those of skill in the art, asexemplified by the technique of Kohler & Milstein (1975), and furtherdescribed hereinbelow.

Generally speaking, a preferred method of preparing MAbs usingstimulated endothelial cells involves the following procedures: Cells orcell lines derived from human tumors are grown in tissue culture for ≧4days. The tissue culture supernatant (`tumor-conditioned medium`) isremoved from the tumor cell cultures and added to cultures of HUVEC at afinal concentration of 50% (v/v). After 2 days culture the HUVEC areharvested non-enzymatically and 1-2×10⁶ cells injected intraperitoneallyinto mice. This process is repeated three times at two-weekly intervals,the final immunization being by the intravenous route. Three days laterthe spleen cells are harvested and fused with SP2/0 myeloma cells bystandard protocols (Kohler & Milstein, 1975) and hybridomas producingantibodies with the appropriate reactivity are cloned by limitingdilution.

From the resultant collection of hybridomas, one will then desire toselect one of more hybridomas that produce an antibody that recognizesthe activated vascular endothelium to a greater extent than itrecognizes non-activated vascular endothelium. One goal is theidentification of antibodies having virtually no binding affinity fornormal endothelium. However, in contrast to the prior art, in thepresent invention this property is not critical. In any event, one willgenerally identify suitable antibody-producing hybridomas by screeningusing, e.g., an ELISA, RIA, IRMA, IIF, or similar immunoassay, againstone or more types of tumor-activated endothelial cells. Once candidateshave been identified, one will desire to test for the absence ofreactivity for non-activated or "normal" endothelium or other normaltissue or cell type. In this manner, hybridomas producing antibodieshaving an undesirably high level of normal cross-reactivity for theparticular application envisioned may be excluded.

(f) Anti-Endoglin Antibodies

Using the technique described above, antibodies having relativespecificity for tumor vascular endothelium have been prepared andisolated. In one particular example, HT29 carcinoma cells were employedto prepare the conditioned medium, which was then employed to stimulateHUVE cells in culture. The resultant HT29-activated HUVE cells were thenemployed as immunogens in the preparation of a hybridoma bank, which wasELISA-screened using HT29-activated HUVE cells and by immunohistologicanalysis of sections of human tumors and normal tissues. From this bank,antibodies that recognized a tumor vascular endothelial cell antigenwere selected.

The MAbs termed tumor endothelial cell antibody 4 and tumor endothelialcell antibody 11 (TEC4 and TEC11) were obtained using the above method.The antigen recognized by TEC4 and TEC11 was ultimately determined to bethe molecule endoglin. The epitopes on endoglin recognized by TEC4 andTEC11 are present on the cell surface of stimulated HUVE cells, and onlyminimally present (or immunologically accessible) on the surface ofnon-stimulated cells. MAbs have previously been raised against endoglin.However, analyzing the reactivity with HUVEC or TCM-activated HUVEC cellsurface determinants by FACS or indirect immunofluorescence shows theepitopes recognized by TEC-4 and TEC-11 to be distinct from those of aprevious antibody termed 44G4 (Gougos & Letarte, 1988).

Although any of the known anti-endoglin antibodies (e.g., Gougos &Letarte, 1988; Gougos et al., 1992; O'Connell et al., 1992; Buhring etal., 1991) may be used in connection with the present invention, theTEC-4 and TEC-11 mAbs are envisioned to be particularly suitable. Thisis because they label capillary and venular endothelial cells moderatelyto strongly in a broad range of solid tumors (and in several chronicinflammatory conditions and fetal placenta), but display relatively weakstaining of vessels in the majority of normal, healthy adult tissues.TEC-11 is particularly preferred as it shows virtually no reactivitywith non-endothelial cells. Furthermore, both TEC-4 and TEC-11 arecomplement-fixing, which imparts to them the potential to also induceselective lysis of endothelial cells in the tumor vascular bed.

Antibodies that are cross-reactive with the MAbs TEC-4 and TEC-11, i.e.,those that bind to endoglin at the same epitope as TEC-4 or TEC-11, arealso contemplated to be of use in this invention. The identification ofan antibody or antibodies that bind to endoglin at the same epitopes asTEC-4 or TEC-11 is a fairly straightforward matter. This can be readilydetermined using any one of variety of immunological screening assays inwhich antibody competition can be assessed. For example, where the testantibodies to be examined are obtained from a different source to thatof TEC-4 or TEC-11, e.g., a rabbit, or are even of a different isotype,for example, IgG1 or IgG3, a competition ELISA may be employed. In onesuch embodiment of a competition ELISA one would pre-mix TEC-4 or TEC-11with varying amounts of the test antibodies prior to applying to theantigen-coated wells in the ELISA plate. By using either anti-murine oranti-IgM secondary antibodies one will be able to detect only the boundTEC-4 or TEC-11 antibodies--the binding of which will be reduced by thepresence of a test antibody that recognizes the same epitope as eitherTEC-4 or TEC-11.

To conduct an antibody competition study between TEC-4 or TEC-11 and anytest antibody, one may first label TEC-4 or TEC-11 with a detectablelabel, such as, e.g., biotin or an enzymatic or radioactive label, toenable subsequent identification. In these cases, one would incubate thelabelled antibodies with the test antibodies to be examined at variousratios (e.g., 1:1, 1:10 and 1:100) and, after a suitable period of time,one would then assay the reactivity of the labelled TEC-4 or TEC-11antibodies and compare this with a control value in which no potentiallycompeting antibody (test) was included in the incubation.

The assay may be any one of a range of immunological assays based uponantibody binding and the TEC-4 or TEC-11 antibodies would be detected bymeans of detecting their label, e.g., using streptavidin in the case ofbiotinylated antibodies or by using a chromogenic substrate inconnection with an enzymatic label or by simply detecting theradiolabel. An antibody that binds to the same epitope as TEC-4 orTEC-11 will be able to effectively compete for binding and thus willsignificantly reduce TEC-4 or TEC-11 binding, as evidenced by areduction in labelled antibody binding. In the present case, aftermixing the labelled TEC-4 or TEC-11 antibodies with the test antibodies,suitable assays to determine the remaining reactivity include, e.g.,ELISAs, RIAs or western blots using human endoglin; immunoprecipitationof endoglin; ELISAs, RIAs or immunofluorescent staining of recombinantcells expressing human endoglin; indirect immunofluorescent staining oftumor vasculature endothelial cells; reactivity with HUVEC orTCM-activated HUVEC cell surface determinants indirectimmunofluorescence and FACS analysis. This latter method is mostpreferred and was employed to show that the epitopes recognized by TEC-4and TEC-11 are distinct from that of 44G4 (Gougos & Letarte, 1988).

The reactivity of the labelled TEC-4 or TEC-11 antibodies in the absenceof any test antibody is the control high value. The control low value isobtained by incubating the labelled antibodies with unlabelledantibodies of the same type, when competition would occur and reducebinding of the labelled antibodies. A significant reduction in labelledantibody reactivity in the presence of a test antibody is indicative ofa test antibody that recognizes the same epitope, i.e., one that"cross-reacts" with the labelled antibody. A "significant reduction" inthis aspect of the present application may be defined as a reproducible(i.e., consistently observed) reduction in binding of at least about10%-50% at a ratio of about 1:1, or more preferably, of equal to orgreater than about 90% at a ratio of about 1:100.

The use of "cross-reactivity assays", as described above in the contextof TEC-4 and TEC-11 antibodies, may be applied to any antibody for usein the present invention. Therefore, antibodies that bind to a componentof a tumor cell, a component of tumor vasculature, a tumorcell-associated component, a tumor vasculature-associated component, atumor extracellular matrix component, or to any cell type listed herein,at the same epitope as any of the antibodies listed herein, asdetermined by an antibody competition assay, will be an antibody thatfalls under the scope of this invention when combined with a coagulatingagent to form a bispecific ligand.

(g) Use of Vascular Endothelial Cell Binding Ligands

Biological ligands that are known to bind or interact with endothelialcell surface molecules, such as growth factor receptors, may also beemployed as a targeting component.

The growth factors or ligands contemplated to be useful as targets inthis sense include VEGF/VPF, FGF, TGFβ, ligands that bind to a TIE,tumor-associated fibronectin isoforms, scatter factor, hepatocyte growthfactor (HGF), platelet factor 4 (PF4), PDGF and TIMP.

Particularly preferred targets are VEGF/VPF, the FGF family of proteinsand TGFβ. Abraham et al. (1986) cloned FGF, which is therefore availableas a recombinant protein. As reported by Ferrara et al. (1991), fourspecies of VEGF having 121, 165, 189, and 206 amino acids have beencloned.

(h) Targeting of Bound Ligands

Antibodies or specific targeting ligands may also be directed to anycomponent that binds to the surface of vascular endothelial cells in adisease site, such as a tumor. Such components are exemplified bytumor-derived ligands and antigens, such as growth factors, that bind tospecific cell surface receptors already present on the endothelialcells, or to receptors that have been induced, or over-expressed, onsuch cells in response to the tumor environment. Tumorvasculature-associated targets may also be termed tumor-derivedendothelial cell binding factors.

A level of specificity required for successful disease targeting will beachieved partly because the local endothelial cells will be induced toexpress, or reveal, receptors that are not present, or areunder-expressed or masked, on normal endothelial cells. With tumors,further specificity will result due to the fact that endothelial cellsin the tumor will capture the tumor-derived factors, and bind them tothe cell surface, reducing the amount of ligand available for othertissues. When combined with the further dilution of the factor or ligandby distribution in the blood and tissue fluid pool, endothelial cells innormal tissues will be expected to bind relatively little of suchfactors. Thus, operationally, cell-surface bound ligands or factors willbe able to used as a tumor endothelial cell marker.

In addition to manufacture by the tumor cells themselves, tumorendothelial cell binding factors may also originate from other celltypes, such as macrophages and mast cells, that have infiltrated tumors,or may be elaborated by platelets that become activated within thetumor.

Further growth factors or ligands contemplated to be useful as tumorvasculature-associated targets include EGF, FGF, VEGF, TGFβ, HGF(NaKamura, 1991), angiotropin, TGFα, TNF-α, PD-ECGF and TIE bindingligands (Bicknell and Harris, 1992). The currently preferred targets areVEGF/VPF, the FGF family of proteins, transforming growth factor-β(TGF-β); TGF-α; tumor necrosis factor-α (TNF-α); angiotropin;platelet-derived endothelial cell growth factor (PD-ECGF); TIE bindingligands; pleiotropin.

Another aspect of the present invention is the use of targetingantibodies, or binding regions therefrom, that are specific for epitopespresent only on ligand-receptor complexes, which epitopes are absentfrom both the individual (free) ligand and the receptor in its unboundform. These antibodies recognize and bind to the unique conformationthat results when a ligand, such as a growth factor, binds to itsreceptor, such as a growth factor receptor, to form a specifically boundcomplex. Such epitopes are not present on the uncomplexed forms of theligands or receptors.

The inventors contemplate that the ligand-receptor complexes to whichthese antibodies bind are present in significantly higher number ontumor-associated endothelial cells than on non-tumor associatedendothelial cells. Such antibodies will therefore be useful as targetingagents and will serve to further increase the specificity of thebispecific coagulants of the invention.

(i) Receptor Constructs

Soluble binding domains of endothelial cell surface receptors are alsocontemplated for use as targeting ligands in the present invention. Thisconcept is generally based upon the well-known sandwich bindingphenomena that has been exploited in a variety of in vitro and in vivobinding protocols. Basically, as the endothelial cells express specificreceptors, the cells bind to and adsorb the corresponding ligands, theligands are then available for binding to further receptor constructsshould they be introduced into the system.

A range of useful endothelial cell receptors has been identified in theforegoing sections, with VEGF/VPF, FGF, TGFβ, TIE-1 and TIE-2 beingparticularly preferred targets. Each of these receptors could bemanipulated to form a soluble binding domain for use as a targetingligand.

4. Disease-Associated Stromal Cell Targets

(a) Extracellular Matrix/Stromal Targets

The usefulness of the basement membrane markers in tumoral pathology wasdescribed by Birembaut et al. (1985). These studies showed that thedistribution of basement membrane (BM) markers, type IV collagen,laminin (LM), heparan sulphate proteoglycan (HSP) and fibronectin (FN)were disrupted in tumoral pathology. Burtin et. al. (1983) alsodescribed alterations of the basement membrane and connective tissueantigens in human metastatic lymph nodes.

A preferred target for use with the invention is RIBS. Ugarova et al.(1993) reported that conformational changes occur in fibrinogen and areelicited by its interaction with the platelet membrane glycoproteinGPIIb-IIIa. The binding of fibrinogen to membrane glycoproteinGPIIb-IIIa on activated platelets leads to platelet aggregation. Thisinteraction results in conformational changes in fibrinogen as evidencedby the expression of receptor-induced binding sites, RIBS, epitopeswhich are expressed by the bound but not the free ligand.

Two RIBS epitopes have been localized by Ugarova et al. (1993). Onesequence resides at γ112-119 and is recognized by MAb 9F9; the second isthe RGDF sequence at Aα 95-98 and is recognized by mAb 155B16. Theseepitopes are also exposed by adsorption of fibrinogen onto a plasticsurface and digestion of the molecule by plasmin. Proteolytic exposureof the epitopes coincides with cleavage of the carboxyl-terminal aspectsof the Aα-chains to form fragment X₂. The inaccessibility of the RGDFsequence at Aα 95-98 in fibrinogen suggests that this sequence does notparticipate in the initial binding of the molecule to GPIIb-IIIa.

Binding of fibrinogen to its receptor alters the conformation of thecarboxyl-terminal aspects of the Aα-chains, exposing the sequences whichreside in the coiled-coil connector segments between the D and E domainsof the molecule, generating the RIBS epitopes. In practical terms, theRIBS sequences are proposed as epitopes for use in targeting with acoaguligand. The MAbs 9F9 and 155B16 may thus be advantageously used, asmay the antibodies described by Zamarron et al. (1991).

(b) Additional Cellular Targets

The present invention has the further advantage that it may be used todirect coagulants to disease-associated vasculature by targeting them tocell types found within the disease region.

Platelets participate in hemostasis and thrombosis by adhering toinjured blood vessel walls and accumulating at the site of injury.Although platelet deposition at sites of blood vessel injury isresponsible for the primary arrest of bleeding under physiologicconditions, it can lead to vascular occlusion with ensuing ischemictissue damage and thrombus embolization under pathologic conditions.

Interactions of platelets with their environment and with each otherrepresent complex processes that are initiated at the cell surface. Thesurface membrane, therefore, provides a reactive interface between theexternal medium, including components of the blood vessel wall andplasma, and the platelet interior.

p-155, a multimeric platelet protein that is expressed on activatedplatelets (Hayward et al., 1991), may be targeted using the invention.Platelets respond to a large number of stimuli by undergoing complexbiochemical and morphological changes. These changes are involved inphysiological processes including adhesion, aggregation, andcoagulation. Platelet activation produces membrane alterations that canbe recognized by monoclonal antibodies. The monoclonal antibody JS-1(Hayward et al., 1991) is one such antibody contemplated for use as partof a coaguligand.

Ligand-induced binding sites (LIBS) are sites expressed on cell surfacereceptors only after ligand binding causes the receptor to change shape,mediate subsequent biological events. These may be seen as counterpartsto RIBS and are also preferred targets for use with the presentinvention.

13 anti-LIBS antibodies have been developed by Frelinger et. al. (1990;1991), any one of which may be used to deliver a coagulant to a diseaseor tumor site in accordance herewith. The murine monoclonal antiplateletantibodies MA-TSPI-1 (directed against human thrombospondin) andMA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against LIBS on humanplatelet glycoprotein IIb/IIIa) of Dewerchin et al. (1991) may also beused, as may RUU 2.41 and LIBS-1 of Heynen et al. (1994); OP-G2 ofTomiyama et al. (1992); and Ab-15.

Many other targets, such as antigens on smooth muscle cells, pericytes,fibroblasts, macrophages and infiltrating lymphocytes and leukocytes mayalso be used.

B. Coagulating Agents

The second arm or element of the bispecific agents of the invention willbe a component that is capable of promoting coagulation. "Coagulationpromoting agents" may be coagulation factors, factors that indirectlystimulate coagulation, or they may be in the form of a second bindingregion that is capable of binding and releasing a coagulation factor orfactor that indirectly stimulates coagulation.

1. Coagulation Factors

A variety of coagulation factors may be used in connection with thepresent invention, as exemplified by the agents set forth below. Where acoagulation factor is covalently linked to a first binding agent, a sitedistinct from its functional coagulating site is used to join themolecules. Appropriate joining regions distinct from the active sites,or functional regions, of the coagulation factors are also described ineach of the following sections.

(a) Tissue Factor

Tissue factor (TF) is one agent capable of initiating blood coagulation.TF is the activator of the extrinsic pathway of blood coagulation and isnot in direct contact with the blood under physiologically normalconditions (Osterud et al., 1986; Nemerson, 1988; Broze, 1992; Ruf &Edington, 1994). In vascular damage or activation by certain cytokinesor endotoxin, however, TF will be exposed to the blood, either by the(sub)endothelial cells (Weiss et al., 1989) or by certain blood cells(Warr et al., 1990). TF will then complex with factor VIIa, which undernormal conditions circulates at low concentrations in the blood(Wildgoose et al., 1992), and the TF/factor VIIa complex will start thecoagulation cascade through the activation of factor X into factor Xa.The cascade will ultimately result in the formation of fibrin.

For this sequence of events to occur, the TF:VIIa complex has to beassociated with a phospholipid surface upon which thecoagulation-initiation complexes with factors IX or X can assemble (Rufet al., 1991; Paborsky et al., 1991; Bach et al., 1986). For thisreason, truncated TF (or tTF), from which the transmembrane andcytoplasmic regions have been removed by truncating the gene, is asoluble protein having one hundred-thousandth of the factor X-activatingactivity of native TF (Ruf et al., 1991).

(b) Clotting Factors

Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIa andderivatives, Factor IX/IXa and derivatives, Factor X/Xa and derivatives,Factor XI/XIa and derivatives, Factor XII/XIIa and derivatives, FactorXIII/XIIIa and derivatives, Factor X activator and Factor V activatormay also be used in the present invention.

(c) Venom Coagulants

Russell's viper venom was shown to contain a coagulant protein byWilliams and Esnouf in 1962. Kisiel (1979) isolated a venom glycoproteinthat activates Factor V; and Di Scipio et al. (1977) showed that aprotease from the venom activates human Factor X. The Factor X activatoris the component contemplated for use in this invention.

Monoclonal antibodies specific for the Factor X activator present inRussell's viper venom have also been produced (e.g., MP1 ofPukrittayakamee et al., 1983), and could be used to deliver the agent toa specific target site within the body.

(d) Prostaglandins and Synthetic Enzymes

Thromboxane A₂ is formed from endoperoxides by the sequential actions ofthe enzymes cyclooxygenase and thromboxane synthetase in plateletmicrosomes. Thromboxane A₂ is readily generated by platelets and is apotent vasoconstrictor, by virtue of its capacity to produce plateletaggregation (Whittle et al., 1981).

Both thromboxane A₂ and active analogues thereof are contemplated foruse in the present invention. A synthetic protocol for generatingthromboxane A₂ is described by Bhagwat et al. (1985). The thromboxane A₂analogues described by Ohuchida et. al. (1981) (especially compound 2)are particularly contemplated for use herewith.

It is possible that thromboxane synthase, and other enzymes thatsynthesize platelet-activating prostaglandins, may also be used as"coagulants" in the present context. Shen and Tai (1986a;b) describemonoclonal antibodies to, and immunoaffinity purification of,thromboxane synthase; and Wang et. al. (1991) report the cDNA for humanthromboxane synthase.

(e) Inhibitors of Fibrinolysis

α2-antiplasmin, or α2-plasmin inhibitor, is a proteinase inhibitornaturally present in human plasma that functions to efficiently inhibitthe lysis of fibrin clots induced by plasminogen activator (Moroi &Aoki, 1976). α2-antiplasmin is a particularly potent inhibitor, and iscontemplated for use in the present invention.

α2-antiplasmin may be purified as first described by Moroi and Aoki(1976). Other purification schemes are also available, such as usingaffinity chromatography on plasminogen-Sepharose, ion-exchangechromatography on DEAE-Sephadex and chromatography onConcanavalin-A-Sepharose; or using affinity chromatography on aSepharose column bearing an elastase-digested plasminogen formulationcontaining the three N-terminal triple-loop structures in the plasminA-chain (LBSI), followed by gel filtration (Wiman & Collen, 1977; Wiman,1980, respectively).

As the cDNA sequence for α2-antiplasmin is available (Tone et al.,1977), a preferred method for α2-antiplasmin production will be viarecombinant expression.

Monoclonal antibodies against α2-antiplasmin are also available that maybe used in the bispecific binding ligand embodiments of the invention.For example, Hattey et al. (1987) described two MAbs againstα2-antiplasmin, MPW2AP and MPW3AP. As each of these MAbs were reportedto react equally well with native α2-antiplasmin, they could both beused to deliver exogenous α2-antiplasmin to a target site or to garnerendogenous α2-antiplasmin and concentrate it within the targeted region.Other antibodies, such as JTPI-2, described by Mimuro and colleagues,could also be used.

2. Agents that Bind Coagulation Factors

Another group of bispecific coagulating ligands of this invention arethose in which the targeting region is not directly linked to acoagulation factor, but is linked to a second binding region that bindsto a coagulating factor.

Where a second binding region is used to bind and deliver a coagulationfactor, the binding region is chosen so that it recognizes a site on thecoagulation factor that does not significantly impair its ability toinduce coagulation. The regions of the coagulation factors suitable forbinding in this manner will generally be the same as those regions thatare suitable for covalent linking to the targeting region, as describedin the previous sections.

However, in that bispecific ligands of this class may be expected torelease the coagulation factor following delivery to the tumor site orregion, there is more flexibility allowed in the regions of thecoagulation factor suitable for binding to a second binding agent orantibody. Another advantage is that bispecific antibodies can bepre-localized before infusion of tTF which may reduce the amount of tTfrequired and hence toxicity.

Suitable second binding regions for use in this manner, will generallybe antigen combining sites of antibodies that have binding specificityfor the coagulation factor, including functional portions of antibodies,such as scFv, Fv, Fab', Fab and F(ab')₂ fragments.

Bispecific binding ligands that contain antibodies, or fragmentsthereof, directed against Tissue Factor, Thrombin, Prekallikein, FactorV/Va, Factor VIII/VIIIa, Factor IX/IXa, Factor X/Xa, Factor XI/XIa,Factor XII/XIIa, Factor XIII/XIIIa, Russell's viper venom, thromboxaneA₂ or α2-antiplasmin are exemplary embodiments of this aspect of theinvention.

C. Linkage Means

The first, targeting region and second, coagulating region will beoperatively linked to allow each region to perform its intended functionwithout significant impairment. Thus, the targeting region is capable ofbinding to the intended target, as selected from the range of tumorenvironment targets, and the coagulating region is capable of directlyor indirectly, e.g., through the release of a bound factor, promotingblood coagulation or clotting.

To assess the targeting region binding function, all that is required isto conduct a binding assay to ensure that the bispecific ligand stillbinds to the targeted component in substantially the same manner as theuncomplexed first binding region. The suitable binding assays are of thetype usually seen in immunological binding assays, where the firsttargeting region is an antibody, and/or other biochemical bindingassays, e.g., those using ¹²⁵ Iodine labeled proteins or otherradiolabeled components, as used to assess ligand-receptor binding, togenerate Scatchard plots, and the like.

The target antigen or component in such assays may be provided in manyforms, including proteins purified from natural or recombinant sources,membrane enriched preparations, intact cells and tissue sections.Generally, where protein compositions are used, they will immobilized ona solid support, such as a microtitre plate, a membrane, or even on acolumn matrix. It is also generally preferred to use a targetcomposition that reflects the physiological target, therefore as thetarget will usually be cell-associated, the use of compositions thatinclude intact cells, including tissues and the cells themselves, isalso preferred.

The various immunological assays available to confirm the functionalbinding of a bispecific complex include, e.g., Western blots, ELISAs,ELISAs using fixed cells, immunohistochemistry, and fluorescentactivated cell sorting (FACS). The execution of all such assays isgenerally known to those of skill in the art, and is further disclosedherein.

Assessing the targeting region binding function of a bispecific compoundin any of the above or other binding assays is a straightforward matter,where the bispecific ligand and the uncomplexed first binding regionwill most usually be run in a parallel assay, under the same conditions,to enable ready comparison. Effective bispecific ligands will bind tothe target without significant impairment, i.e., in substantially thesame manner as the uncomplexed first binding region. Taking theuncomplexed binding region assay result as the 100% reference value,"substantial binding" of the bispecific ligand, as used herein, meansthat the bispecific ligand exhibits at least about 50% binding, and morepreferably, between about 50% and about 80% binding, and mostpreferably, between about 80% and about 100% binding.

Where the bispecific ligand includes a second binding region that bindsto a coagulant, e.g., it is a bispecific antibody, further useful assaysare those of the type that allow the binding functions of both arms ofthe bispecific ligand to be assessed at the same time. For example, thismay be achieved by assessing the binding of a radiolabeled coagulant toa target cell via bridging with the bispecific ligand or antibody. Suchan assay is exemplified by the binding of tTF to target cells using theB21-2/10H10 bispecific antibody, as described in Example II.

Determining the coagulating agent function of the bispecific ligand isalso a straightforward matter. All that is required here is to conduct acoagulation assay using the bispecific ligand and ensure that itfunctions to promote coagulation in substantially the same manner as theuncomplexed coagulating agent. This is true for "coagulating agents"that are both coagulation factors themselves and those that are secondbinding regions that bind to a coagulation factor. Naturally, in thelatter case, in an in vitro or ex vivo assay, the bispecific ligand willbe precomplexed with the coagulation factor to allow binding to thesecond binding region.

One suitable coagulation assay is that in which the bispecific ligands,pre-complexed with coagulant if necessary, are admixed with a plasmasample. The appearance of fibrin strands is indicative of coagulation inthis assay. Effective bispecific ligands would thus be expected toreduce the time taken for fibrin strands to appear, and particularly, tosignificantly reduce the elapsed time in comparison to control levels.

A variation of the above assay involves first exposing appropriatetarget cells to the bispecific ligand under conditions effective, andfor a time sufficient, to allow binding, washing the cells to removenon-specifically bound components and then resuspending the washed cellsin plasma. Only cells effectively coated with the bispecific ligandwould be expected to reduce the time taken for fibrin strands to appearin the assay. This type of assay is preferred in that it is, in itself,an assay that assesses both of the functions of the bispecificconstruct, i.e., initial targeting to the cell and subsequent localizedcoagulation.

To compare the coagulating function of a bispecific compound to that ofan uncomplexed coagulating agent, parallel assays may again beconducted. Effective bispecific ligands will function to promotecoagulation without significant impairment, i.e., will function insubstantially the same manner as the uncomplexed coagulating agent.Taking the uncomplexed coagulant assay result as the 100% referencevalue, "substantial function", as used herein, means that the bispecificligand exhibits at least about 50% coagulation, and more preferably,between about 50% and about 80% coagulation, and most preferably,between about 80% and about 100% coagulation.

The two functional regions of the bispecific ligands may be joined usingsynthetic chemistry techniques or recombinant DNA techniques. Each ofthese techniques are routinely employed and well known to those of skillin the art, and are further exemplified in Example I and by the detailsset forth below.

1. Biochemical Cross-linkers

The joining of an antibody, or other targeting component, to acoagulating agent will generally employ the same technology as developedfor the preparation of immunotoxins. However, considerable advantagesare apparent in the present technology, as the consequences of a certainamount of uncomplexed coagulating agent becoming availablephysiologically are not contemplated to be particularly severe. Thus,the stability requirements for any cross-linkers are not so stringent asfor linkers employed in other constructs, such as immunotoxins.Therefore, it can be considered as a general guideline that anybiochemical crosslinker that is appropriate for use in an immunotoxinwill also be of use in the present context, and additional linkers mayalso be considered.

In addition to toxins, a variety of other chemotherapeutic andpharmacological agents have been linked to antibodies to form conjugatesthat have been shown to function pharmacologically (see, e.g., Vaickuset al., 1991). Exemplary antineoplastic agents that have beeninvestigated include doxorubicin, daunomycin, methotrexate andvinblastine, amongst others (Dillman et al., 1988; Pietersz et al.,1988). Moreover, the attachment of other agents such as neocarzinostatin(Kimura et al., 1983), macromycin (Manabe et al., 1984), trenimon(Ghose, 1982) and α-amanitin (Davis & Preston, 1981) has been described.The linking technology described in each of the foregoing scientificpapers is also contemplated for use in connection with the presentinvention.

Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different molecules, e.g., a bindingand coagulating agent. To link two different proteins in a step-wisemanner, heterobifunctional cross-linkers can be used that eliminateunwanted homopolymer formation (Table VI).

                                      TABLE VI                                    __________________________________________________________________________    HETEROBIFUNCTIONAL CROSS-LINKERS                                                                              Spacer Arm                                                                                   Length\after         linker       Reactive Toward                                                                        Advantages and Applications                                                                    cross-linking                          __________________________________________________________________________     SMPT   Primary amines                                                                        Greater stability                                                                            11.2 Å                                                             Sulfhydryls                                           SPDP               Thiolationines                                                                                                          6.8 Å                               Cleavable cross-linking                                LC-SPDP         Primary amines                                                                  Extended spacer arm                                                                                              15.6 Å                                        Sulfhydryls                                          Sulfo-LC-SPDP                                                                           Primary amines                                                                        Extended spacer arm                                                                                             15.6 Å                                       Water-soluble                                          SMCC              Stable maleimide reactive group                                                                    11.6 Å                                                    Enzyme-antibody conjugation                                                            Hapten-carrier protein conjugation            Sulfo-SMCC                                                                                 Primary amines                                                                      Stable maleimide reactive group                                                                    11.6 Å                                                   Water-soluble                                                                               Enzyme-antibody conjugation              MBS               Enzyme-antibody conjugation                                                                              9.9 Å                                               Hapten-carrier protein conjugation                     Sulfo-MBS                                                                                   Primary amines                                                                    Water-soluble                                                                                                           9.9 Å                                  Sulfhydryls                                          SIAB              Enzyme-antibody conjugation                                                                             10.6 Å                                                 Sulfhydryls                                          Sulfo-SIAB                                                                                 Primary amines                                                                      Water-soluble                                                                                                         10.6 Å                                  Sulfhydryls                                          SMPB              Extended spacer arm                                                                                             14.5 Å                                    Enzyme-antibody conjugation                               Sulfo-SMPB                                                                                 Primary amines                                                                     Extended spacer arm                                                                                             14.5 Å                                       Water-soluble                                          EDC/Sulfo-NHS                                                                           Primary amines                                                                        Hapten-Carrier conjugation                                                                                  0                                                      Carboxyl groups                                      ABH                  Reacts with sugar groups                                                                                 11.9 Å                                             Nonselective                                         __________________________________________________________________________

An exemplary heterobifunctional cross-linker contains two reactivegroups: one reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or fragment) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the coagulant).

It can therefore be seen that the preferred coagulants or coagulantbinding regions will generally have, or be derivatized to have, afunctional group available for cross-linking purposes. This requirementis not considered to be limiting in that a wide variety of groups can beused in this manner. For example, primary or secondary amine groups,hydrazide or hydrazine groups, carboxyl alcohol, phosphate, oralkylating groups may be used for binding or cross-linking. For ageneral overview of linking technology, one may wish to refer to Ghose &Blair (1987).

The spacer arm between the two reactive groups of a cross-linkers mayhave various length and chemical compositions. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents). The use of peptide spacers, such asL-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andcoagulating agents. Linkers that contain a disulfide bond that issterically hindered may prove to give greater stability in vivo,preventing release of the coagulant prior to binding at the site ofaction. These linkers are thus one preferred group of linking agents.

One of the most preferred cross-linking reagents for use in immunotoxinsis SMPT, which is a bifunctional cross-linker containing a disulfidebond that is "sterically hindered" by an adjacent benzene ring andmethyl groups. It is believed that stearic hindrance of the disulfidebond serves a function of protecting the bond from attack by thiolateanions such as glutathione which can be present in tissues and blood,and thereby help in preventing decoupling of the conjugate prior to thedelivery of the attached agent to the tumor site. It is contemplatedthat the SMPT agent may also be used in connection with the bispecificcoagulating ligands of this invention.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes theheterobifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers can also beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of suchcross-linkers is well understood in the art.

Once conjugated, the bispecific agent will generally be purified toseparate the conjugate from unconjugated targeting agents or coagulantsand from other contaminants. It is important to remove unconjugatedtargeting agent to avoid the possibility of competition for the antigenbetween conjugated and unconjugated species. A large a number ofpurification techniques are available for use in providing conjugates ofa sufficient degree of purity to render them clinically useful.Purification methods based upon size separation, such as gel filtration,gel permeation or high performance liquid chromatography, will generallybe of most use. Other chromatographic techniques, such as Blue-Sepharoseseparation, may also be used.

2. Recombinant Fusion Proteins

The bispecific targeted coagulants of the invention may also be fusionproteins prepared by molecular biological techniques. The use ofrecombinant DNA techniques to achieve such ends is now standard practiceto those of skill in the art. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. DNA and RNA synthesis may,additionally, be performed using an automated synthesizers (see, forexample, the techniques described in Sambrook et al., 1989; and Ausubelet al., 1989).

In general, to prepare a fusion a protein one would join a DNA codingregion, such as a gene or cDNA, encoding a binding ligand or othertargeting region to a DNA coding region (i.e., gene or cDNA) encoding acoagulation factor or coagulant binding region. This typically involvespreparing an expression vector that comprises, in the same readingframe, a first DNA segment encoding the first binding region operativelylinked to a second DNA segment encoding the coagulation factor. Thesequences are attached in a manner such that translation of the totalnucleic acid yields the desired bispecific compounds of the invention.Expression vectors contain one or more promoters upstream of theinserted DNA regions that act to promote transcription of the DNA and tothus promote expression of the encoded recombinant protein. This is themeaning of "recombinant expression".

Should a particular binding region or coagulant be preferred, and theencoding DNA not instantly available, it may be obtained using thetechniques of "molecular cloning" in which a DNA molecule encoding thedesired protein is obtained from a DNA library (e.g., a cDNA or genomiclibrary). In such procedures, an appropriate DNA library is screened,e.g., using an expression screening protocol employing antibodiesdirected against the protein, or activity assays. Alternatively,screening may be based on the hybridization of oligonucleotide probes,designed from a consideration of portions of the amino acid sequence ofthe protein, or from the DNA sequences of genes encoding relatedproteins. The operation of such screening protocols are well known tothose of skill in the art and are described in detail in the scientificliterature, for example, in Sambrook et al. (1989).

When produced via recombinant DNA techniques, the targetingagent/coagulating agent compounds of the invention are referred to as"fusion proteins". It is to be understood that such fusion proteinscontain, at least, a targeting agent and a coagulating agent as definedin this invention, and that the agents are operatively attached. Thefusion proteins may also include additional peptide sequences, such aspeptide spacers which operatively attach the targeting agent andcoagulating agent compounds, as long as such additional sequences do notappreciably affect the targeting or coagulating activities of theresultant fusion protein.

It will be understood that the recombinant bispecific protein ligandsmay differ from those bispecific constructs generated by chemicallycross-linking the so-called naturally-produced proteins. In particular,the degree of post-translational modifications, such as, for example,glycosylation and phosphorylation may be different between recombinantfusions and chemical fusions of the same two proteins. This is notcontemplated to be a significant problem, however, those of skill in theart will know to confirm that a recombinant fusion protein functions asintended, and expected from other data, before use in a clinicalsetting.

One advantage of recombinant expression is that the linking regions canbe readily manipulated so that, e.g., their length and/or amino acidcomposition is readily variable. Non-cleavable peptide spacers may beprovided to operatively attach the two agents of the invention, ifdesired. Equally, peptides with unique cleavage sites could be insertedbetween the two components.

If desired in a specific instance, it is possible to provide a peptidespacer operatively attaching the targeting agent and coagulating agentwhich is capable of folding into a disulfide-bonded loop structure.Proteolytic cleavage within the loop would then yield a heterodimericpolypeptide wherein the targeting agent and the coagulating agent arelinked by only a single disulfide bond (see, for example, Lord et al.,1992).

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein expression in a variety of host-expression systems. The celltypes available for expression include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing targeting agent/coagulant coding sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing targeting agent/coagulating agent codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the targetingagent/coagulating agent coding sequences; plant cell systems infectedwith recombinant virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing the targetingagent/coagulant coding sequence; and mammalian cell systems (e.g., COS,CHO, BHK, 293, 3T3) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for thetargeting agent/coagulating agent construct being expressed. Forexample, when large quantities of bispecific agent are to be produced,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983), in which the targeting agent/coagulating agentcoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein additionallycontaining a portion of the lac Z product is provided; pIN vectors(Inouye et al., 1985; Van Heeke et al., 1989); and the like. PGEXvectors may also be used to express foreign polypeptides, such as thetargeting agent/coagulating agent combinations as fusion proteinsadditionally containing glutathione S-transferase (GST). In general,such fusion proteins are soluble and can easily be purified from lysedcells by adsorption to glutathione-agarose beads followed by elution inthe presence of free glutathione. The PGEX vectors are designed toinclude thrombin or factor Xa protease cleavage sites so that thebinding agent/coagulant protein of the overall fusion protein can bereleased from the GST moiety.

In a useful insect system, Autograph californica nuclear polyhidrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The targeting agent/coagulatingagent coding sequences may be cloned into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of the bispecific ligand coding sequences will result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted gene isexpressed (e.g., see Smith et al., 1983; *U.S. Pat. No. 4,215,051,Smith).

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the targeting agent/coagulating agent coding sequences may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing bispecific proteins in infectedhosts (e.g., see Logan et al., 1984).

Specific initiation signals may also be required for efficienttranslation of inserted targeting agent/coagulating agent codingsequences. These signals include the ATG initiation codon and adjacentsequences. Exogenous translational control signals, including the ATGinitiation codon, may additionally need to be provided. One of ordinaryskill in the art would readily be capable of determining this andproviding the necessary signals. It is well known that the initiationcodon must be in phase (or in-frame) with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe of a variety of origins, both natural and synthetic. The efficiencyof expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., 1987).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expressconstructs encoding the targeting agent/coagulant ligands may beengineered. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with targetingagent/coagulant DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limited,to the herpes simplex virus thymidine kinase (Wigler et al., 1977),hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., 1962),and adenine phosphoribosyltransferase genes (Lowy et al., 1980) can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980;O'Hare et al., 1981); gpt, which confers resistance to mycophenolic acid(Mulligan et al., 1981); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984).

D. Antibodies

Where antibodies are used as one or both portions of the bispecificligand, the choice of antibody will generally be dependent on the typetumor and coagulating ligand chosen. However, certain advantages may beachieved through the application of particular types of antibodies. Forexample, while IgG based antibodies may be expected to exhibit betterbinding capability and slower blood clearance than their Fab'counterparts, Fab' fragment-based compositions will generally exhibitbetter tissue penetrating capability.

1. Monoclonal Antibodies

Means for preparing and characterizing antibodies are well known in theart (See, e.g., *Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988).

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention,either with or without prior immunotolerizing, depending on the antigencomposition and protocol being employed (e.g., tolerizing to a normalcell population and then immunizing with a tumor cell population), andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster injection, may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired titer level is obtained, the immunizedanimal can be bled and the serum isolated and stored, and/or the animalcan be used to generate MAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in *U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified tumor cell or vascular endothelial cell protein,polypeptide, peptide, or intact cell composition. The immunizingcomposition is administered in a manner effective to stimulate antibodyproducing cells. Rodents such as mice and rats are preferred animals,however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (*Goding, 1986, pp. 60-61), butmice are preferred, with the BALB/c mouse being most preferred as thisis most routinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (*Goding, pp. 65-66, 1986; *Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F, 4B210 or one of the above listed mouse cell lines;and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6, are all useful inconnection with human cell fusions.

One preferred murine myeloma cell is the A63-A68, 653 myeloma cell line,which is readily available from the ATCC. Another mouse myeloma cellline that may be used is the 8-azaguanine-resistant mouse murine myelomaSP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 4:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler & Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods is also appropriate (*Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10³¹ 8. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines could also be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

The inventors also contemplate the use of a molecular cloning approachto generate monoclonals. For this, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and control cellse.g., normal-versus-tumor cells. The advantages of this approach overconventional hybridoma techniques are that approximately 10⁴ times asmany antibodies can be produced and screened in a single round, and thatnew specificities are generated by H and L chain combination whichfurther increases the chance of finding appropriate antibodies.

Where MAbs are employed in the present invention, they may be of human,murine, monkey, rat, hamster, chicken or even rabbit origin. Theinvention contemplates the use of human antibodies, "humanized" orchimeric antibodies from mouse, rat, or other species, bearing humanconstant and/or variable region domains, and other recombinantantibodies and fragments thereof. Of course, due to the ease ofpreparation and ready availability of reagents, murine monoclonalantibodies will typically be preferred.

2. Functional Antibody Binding Regions

The origin or derivation of the targeting agent antibody or antibodyfragment (e.g., Fab', Fab, F(ab')₂, Fv or scFv) is not believed to beparticularly crucial to the practice of the invention, so long as theantibody or fragment that is actually employed for the preparation ofthe bispecific ligand exhibits the desired binding properties.

It may be necessary to use antibody preparations in which the Fc portionhas been removed. Fragmentation of immunoglobulin molecules can beachieved by controlled proteolysis, although the conditions will varyconsiderably with species and immunoglobulin class or subclass. BivalentF(ab')₂ fragments are usually preferable over the univalent Fab or Fab'fragments.

Fab

Fab fragments can be obtained by proteolysis of the whole immunoglobulinby the non-specific thiol protease, papain. Papain must first beactivated by reducing the sulphydryl group in the active site withcysteine, 2-mercaptoethanol or dithiothreitol. Heavy metals in the stockenzyme should be removed by chelation with EDTA (2 mM) to ensure maximumenzyme activity. Enzyme and substrate are normally mixed together in theratio of 1:100 by weight. After incubation, the reaction can be stoppedby irreversible alkylation of the thiol group with iodoacetamide orsimply by dialysis. The completeness of the digestion should bemonitored by SDS-PAGE and the various fractions separated by proteinA-Sepharose or ion exchange chromatography.

F(ab')₂

The usual procedure for preparation of F(ab')₂ fragments from IgG ofrabbit and human origin is limited proteolysis by the enzyme pepsin(Protocol 7.3.2). The conditions, 100x antibody excess w/w in acetatebuffer at pH 4.5, 37° C., suggest that antibody is cleaved at theC-terminal side of the inter-heavy-chain disulfide bond. Rates ofdigestion of mouse IgG may vary with subclass and it may be difficult toobtain high yields of active F(ab')₂ fragments without some undigestedor completely degraded IgG. In particular, IgG_(2b) is highlysusceptible to complete degradation. The other subclasses requiredifferent incubation conditions to produce optimal results.

Digestion of rat IgG by pepsin requires conditions including dialysis in0.1 M acetate buffer, pH 4.5, and then incubation for four hours with 1%w/w pepsin; IgG₁ and IgG_(2a), digestion is improved if first dialysedagainst 0.1 M formate buffer, pH 2.8, at 4° C., for 16 hours followed byacetate buffer. IgG_(2b) gives more consistent results with incubationin staphylococcal V8 protease (3% w/w) in 0.1 M sodium phosphate buffer,pH 7.8, for four hours at 37° C.

3. Bispecific Antibodies

In general, the preparation of bispecific antibodies is also well knownin the art, as exemplified by Glennie et al. (1987). Bispecificantibodies have been employed clinically, for example, to treat cancerpatients (Bauer et al., 1991). One method for the preparation ofbispecific antibodies involves the separate preparation of antibodieshaving specificity for the targeted tumor cell antigen, on the one hand,and the coagulating agent (or other desired target, such as anactivating antigen) on the other.

Bispecific antibodies have also been developed particularly for use asimmunotherapeutic agents. As mentioned earlier in conjunction withantigen-induction, certain of these antibodies were developed tocross-link lymphocytes and tumor antigens (Nelson, 1991; Segal et al.,1992). Examples include chimeric molecules that bind T cells, e.g., atCD3, and tumor antigens, and trigger lymphocyte-activation by physicallycross-linking the TCR/CD3 complex in close proximity to the target cell(Staerz et al., 1985; Perez et al., 1985; 1986a; 1986b; Ting et al.,1988).

Indeed, tumor cells of carcinomas, lymphomas, leukemias and melanomashave been reported to be susceptible to bispecific antibody-mediatedkilling by T cells (Nelson, 1991; Segal et al., 1992; deLeij et al.,1991). These type of bispecific antibodies have also been used inseveral Phase I clinical trials against diverse tumor targets. Althoughthey are not novel compositions in accordance with this invention, thecombined use of bispecific cross-linking antibodies along with thebispecific coagulating ligands described herein is also contemplated.The bispecific cross-linking antibodies may be administered as describedin references such as deLeij et al. (1991); Clark et al. (1991);Rivoltini et al. (1992); Bolhuis et al. (1992); and Nitta et al. (1990).

While numerous methods are known in the art for the preparation ofbispecific antibodies, the Glennie et al. (1987) method involves thepreparation of peptic F(ab'γ)₂ fragments from the two chosen antibodies,followed by reduction of each to provide separate Fab'γSH fragments. TheSH groups on one of the two partners to be coupled are then alkylatedwith a cross-linking reagent such as o-phenylenedimaleimide to providefree maleimide groups on one partner. This partner may then beconjugated to the other by means of a thioether linkage, to give thedesired F(ab'γ)₂ heteroconjugate.

Due to ease of preparation, high yield and reproducibility, the Glennieet al. (1987) method is often preferred for the preparation ofbispecific antibodies, however, there are numerous other approaches thatcan be employed and that are envisioned by the inventors. For example,other techniques are known wherein crosslinking with SPDP or protein Ais carried out, or a trispecific construct is prepared (Titus et al.,1987; Tutt et al., 1991).

Another method for producing bispecific antibodies is by the fusion oftwo hybridomas to form a quadroma (Flavell et al., 1991, 1992; Pimm etal., 1992; French et al., 1991; Embleton et al., 1991). As used herein,the term "quadroma" is used to describe the productive fusion of two Bcell hybridomas. Using now standard techniques, two antibody producinghybridomas are fused to give daughter cells, and those cells that havemaintained the expression of both sets of clonotype immunoglobulin genesare then selected.

A preferred method of generating a quadroma involves the selection of anenzyme deficient mutant of at least one of the parental hybridomas. Thisfirst mutant hybridoma cell line is then fused to cells of a secondhybridoma that had been lethally exposed, e.g., to iodoacetamide,precluding its continued survival. Cell fusion allows for the rescue ofthe first hybridoma by acquiring the gene for its enzyme deficiency fromthe lethally treated hybridoma, and the rescue of the second hybridomathrough fusion to the first hybridoma. Preferred, but not required, isthe fusion of immunoglobulins of the same isotype, but of a differentsubclass. A mixed subclass antibody permits the use if an alternativeassay for the isolation of a preferred quadroma.

In more detail, one method of quadroma development and screeninginvolves obtaining a hybridoma line that secretes the first chosen MAband making this deficient for the essential metabolic enzyme,hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtaindeficient mutants of the hybridoma, cells are grown in the presence ofincreasing concentrations of 8-azaguanine (1×10⁻⁷ M to 1×10⁻⁵ M). Themutants are subcloned by limiting dilution and tested for theirhypoxanthine/aminopterin/thymidine (HAT) sensitivity. The culture mediummay consist of, for example, DMEM supplemented with 10% FCS, 2 mML-Glutamine and 1 mM penicillin-streptomycin.

A complementary hybridoma cell line that produces the second desired MAbis used to generate the quadromas by standard cell fusion techniques(Galfre et al., 1981), or by using the protocol described by Clark etal. (1988). Briefly, 4.5×10⁷ HAT-sensitive first cells are mixed with2.8×10⁷ HAT-resistant second cells that have been pre-treated with alethal dose of the irreversible biochemical inhibitor iodoacetamide (5mM in phosphate buffered saline) for 30 minutes on ice before fusion.Cell fusion is induced using polyethylene glycol (PEG) and the cells areplated out in 96 well microculture plates. Quadromas are selected usingHAT-containing medium. Bispecific antibody-containing cultures areidentified using, for example, a solid phase isotype-specific ELISA andisotype-specific immunofluorescence staining.

In one identification embodiment to identify the bispecific antibody,the wells of microtiter plates (Falcon, Becton Dickinson Labware) arecoated with a reagent that specifically interacts with one of the parenthybridoma antibodies and that lacks cross-reactivity with bothantibodies. The plates are washed, blocked, and the supernatants (SNs)to be tested are added to each well. Plates are incubated at roomtemperature for 2 hours, the supernatants discarded, the plates washed,and diluted alkaline phosphatase-anti-antibody conjugate added for 2hours at room temperature. The plates are washed and a phosphatasesubstrate, e.g., P-Nitrophenyl phosphate (Sigma, St. Louis) is added toeach well. Plates are incubated, 3N NaOH is added to each well to stopthe reaction, and the OD₄₁₀ values determined using an ELISA reader.

In another identification embodiment, microtiter plates pretreated withpoly-L-lysine are used to bind one of the target cells to each well, thecells are then fixed, e.g. using 1% glutaraldehyde, and the bispecificantibodies are tested for their ability to bind to the intact cell. Inaddition, FACS, immunofluorescence staining, idiotype specificantibodies, antigen binding competition assays, and other methods commonin the art of antibody characterization may be used in conjunction withthe present invention to identify preferred quadromas.

Following the isolation of the quadroma, the bispecific antibodies arepurified away from other cell products. This may be accomplished by avariety of protein isolation procedures, known to those skilled in theart of immunoglobulin purification. Means for preparing andcharacterizing antibodies are well known in the art (See, e.g.,*Antibodies: A Laboratory Manual, 1988).

For example, supernatants from selected quadromas are passed overprotein A or protein G sepharose columns to bind IgG (depending on theisotype). The bound antibodies are then eluted with, e.g. a pH 5.0citrate buffer. The elute fractions containing the BsAbs, are dialyzedagainst an isotonic buffer. Alternatively, the eluate is also passedover an anti-immunoglobulin-sepharose column. The BsAb is then elutedwith 3.5 M magnesium chloride. BsAbs purified in this way are thentested for binding activity by, e.g., an isotype-specific ELISA andimmunofluorescence staining assay of the target cells, as describedabove.

Purified BsAbs and parental antibodies may also be characterized andisolated by SDS-PAGE electrophoresis, followed by staining with silveror Coomassie. This is possible when one of the parental antibodies has ahigher molecular weight than the other, wherein the band of the BsAbsmigrates midway between that of the two parental antibodies. Reductionof the samples verifies the presence of heavy chains with two differentapparent molecular weights.

Furthermore, recombinant technology is now available for the preparationof antibodies in general, allowing the preparation of recombinantantibody genes encoding an antibody having the desired dual specificity(Van Duk et al., 1989). Thus, after selecting the monoclonal antibodieshaving the most preferred binding characteristics, the respective genesfor these antibodies can be isolated, e.g., by immunological screeningof a phage expression library (Oi & Morrison, 1986; Winter & Milstein,1991). Then, through rearrangement of Fab coding domains, theappropriate chimeric construct can be readily obtained.

E. Binding Assays

Although the present invention has significant utility in animal andhuman treatment regimens, it also has many other practical uses. Theseuses are generally related to the specific binding ability of thebispecific compounds. In that all the compounds of the invention includeat least one targeting and binding component, e.g., an antibody, ligand,receptor, or such like, the resultant bispecific construct may be usedin virtually all of the binding embodiments that the original antibody,ligand or receptor, etc., may be used. The presence of the coagulant, orother binding regions, does not negate the utility of the first bindingregions in any binding assay.

As such, the bispecific coagulating ligands may be employed in standardbinding assays, such as in immunoblots, Western blots, and other assaysin which an antigen is immobilized onto a solid support matrix, e.g.,nitrocellulose, nylon or a combination thereof. They may be employedsimply as an "antibody substitute" or may be used to provide amore-specific detection means for use in detecting antigens againstwhich standard secondary reagents cause an unacceptably high background.This is especially useful when the antigens studied are themselvesimmunoglobulins or other antibodies are used in the procedure, asexemplified below in the case of ELISAs.

The bispecific binding ligands may also be used in conjunction with bothfresh-frozen and formalin-fixed, paraffin-embedded tissue blocks inimmunohistochemistry; in fluorescent activated cell sorting, flowcytometry or flow microfluorometry; in immunoprecipitation to separate atarget antigen from a complex mixture, in which case, due to theirpotential to form molecular lattices, they may even achieveprecipitation without a secondary matrix-coupled reagent; in antigen orcell purification embodiments, such as affinity chromatography, evenincluding, in certain cases, the one-step rapid purification of one ormore cell populations at the same time; and in many other binding assaysthat will be known to those of skill in the art given the informationpresented herein.

As an example, the bispecific ligands of the invention may be used inELISA assays. Many types of ELISAs are known and routinely practiced inthe art. The bispecific ligands may be employed in any of the bindingsteps, depending on the particular type of ELISA being performed and the"antigen" (component) to be detected. The ligands could therefore beused to coat the plate, to compete for binding sites, as an antigen toprovide a standard curve, as a primary binding ligand, as a secondarybinding ligand or even as a tertiary or other binding ligand. The manymodes of conducting ELISAs will be known to those of skill in the art,in further light of the exemplary mode discussed below.

In one form of an ELISA, binding targets, generally antibodiesthemselves, are immobilized onto a selected surface, preferably asurface exhibiting a protein affinity such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbedmaterial, it is desirable to bind or coat the assay plate wells with anonspecific protein that is known to be antigenically neutral withregard to the test antisera such as bovine serum albumin (BSA), caseinor solutions of milk powder. This allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

In these types of ELISAs, generally termed sandwich ELISAs, theplate-bound antibody is used to "trap" the antigen. After binding of thefirst antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with, in the present exemplaryembodiment, a test sample containing the antigenic material to bedetected and/or titered in a manner conducive to immune complex(antigen/antibody) formation. These embodiments are particularly usefulfor detecting ligands in clinical samples or biological extracts. Thesamples are preferably diluted with solutions of BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS) and a detergent, e.g.Tween.

The layered antisera is then allowed to incubate for from 2 to 4 hours,at temperatures preferably on the order of 25° to 37° C. Followingincubation, the antisera-contacted surface is washed so as to removenon-immunocomplexed material. A preferred washing procedure includeswashing with a solution such as PBS/Tween, or borate buffer.

Following formation of specific immunocomplexes between the boundantigen and the test sample, and subsequent washing, the occurrence andamount of immunocomplex formation may be determined by subjecting samecomplex to a secondary specific binding component, which is generally anantibody-based component. In a particular embodiment, the bispecificligands of the invention are proposed for use in this step. Furtherspecific binding and washing steps are then conducted.

To provide a detecting means, in the present exemplary embodiment, athird antibody is used that is linked to a detectable label, such as anassociated enzyme that will generate a color development upon incubatingwith an appropriate chromogenic substrate. The third, or tertiary,labeled antibody has binding affinity for a component of the bispecificligand. The ultimate immunocomplex is determined, after appropriatebinding and washing steps, by detecting the label, e.g., by incubatingwith a chromogenic substrate, such as urea and bromocresol purple or2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H₂ O₂.Quantification is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectra spectrophotometer.

Using a bispecific coagulating ligand as a secondary detection reagentin conjunction with the type of ELISA described above has distinctadvantages. For example, it allows the use of a tertiary, labeledantibody that is specific for a portion of the bispecific ligand that isdistinct from the typical antibody constant regions usually targeted. Inparticular, a tertiary binding ligand that is specific for the coagulantportion (or coagulant binding region) of the bispecific construct may beemployed. This novel means of detecting immune complex formation impartsimproved specificity, which is particularly useful in sandwich ELISAswhere the tertiary antibody may cross-react with, and bind to, theoriginal material used to coat the plate, i.e., the original antibody,rather than just binding to the intended secondary antibody. Bydirecting the labelled tertiary component to an non-antibody portion, oreven to a novel antigen combining region, of a bispecific ligand, theproblem of non-specific binding, and unusually high background, will beavoided.

Further practical uses of the bispecific ligands are evident byexploiting their coagulating ability. As all of the proposed compoundsare capable of inducing coagulation, they may be employed, e.g., as acontrol, in any assay that involves coagulation as a component. Thepresence of the targeting component does not negate the utility of thecoagulant in such assays, as each component functions independently ofthe other.

F. Effective Use of Tissue Factor-Binding Bispecific Antibodies

As mentioned earlier, tissue factor (TF) is one agent capable ofinitiating blood coagulation. TF is exposed to the blood in vasculardamage or following activation by certain cytokines. Available TF thencomplexes with factor VIIa to initiate the coagulation cascade thatultimately results in fibrin formation.

In one exemplary embodiment, the inventors have synthesized a bispecificantibody with specificity for antigens on tumor vasculature endothelialcells on one antigen combining site and specificity for theextracellular domains of human TF on the other antigen combining site.The antibody with specificity for human TF was previously shown to bindTF with high affinity without interfering with the factor VIIacomplexing event or the TF/VIIa activity (Morrissey et al., 1988).Instead of using full length human TF, the inventors used a truncatedform (tTF), which is devoid of the cytoplasmic as well as thetransmembrane domain. Truncated TF lacks coagulation inducing activity,while still being able to complex factor VIIa, probably because it isnot able to complex with a membrane surface upon which thecoagulation-initiation complexes, including Factor X, could assemble.

The mouse model used for analyzing the effectiveness of this tumorvasculature endothelial cell specific targeting construct was a recentlyestablished model in which MHC class II antigens, that are absent fromthe vasculature of normal tissues, are expressed on the tumorvasculature through induction by IFN-γ that is secreted by the tumorcells (Burrows et al., 1992; Burrows & Thorpe, 1993). It has beendemonstrated that anti-class II antibody administered intravenouslylocalizes rapidly and strongly to the tumor vasculature (Burrows et al.,1992).

The present inventors herein demonstrate that, in a C1300 (Muγ) tumorbearing mouse, the anti-MHC Class II/anti-TF bispecific antibody is ableto induce coagulation specifically in the vasculature of the tumor whenadministered together with tTF. Indeed, intravenous administration ofthe antibody:tTF complex induced rapid thrombosis of tumor vasculatureand complete tumor regressions in 70% of animals. Neither the bispecificantibody alone, nor tTF alone, nor any of the isotype matched controlantibodies in the presence or absence of tTF, was able to elicit thesame effect. This indicates that the B21-2/10H10 bispecific antibodyacts as a "coaguligand" that is capable of bridging target cells and tTFso that tTF can activate factor X and start the coagulation cascade. Italso shows the evident success of the coaguligand in treating solidtumors.

G. Pharmaceutical Compositions and Kits

Pharmaceutical compositions of the present invention will generallycomprise an effective amount of the bispecific coagulating liganddissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium.

The phrases "pharmaceutically or pharmacologically acceptable" refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. As used herein, "pharmaceutically acceptablecarrier" includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

1. Parenteral Formulations

The bispecific ligands of the present invention will often be formulatedfor parenteral administration, e.g., formulated for injection via theintravenous, intramuscular, sub-cutaneous or other such routes,including direct instillation into a tumor or disease site. Thepreparation of an aqueous composition that contains a tumor-targetedcoagulant agent as an active ingredient will be known to those of skillin the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

The bispecific ligands or antibodies can be formulated into acomposition in a neutral or salt form. Pharmaceutically acceptablesalts, include the acid addition salts (formed with the free aminogroups of the protein) and which are formed with inorganic acids suchas, for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric, mandelic, and the like. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus nyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. Formulations are easily administered in a variety of dosageforms, such as the type of injectable solutions described above, butdrug release capsules and the like can also be employed.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the bispecific ligand admixed withan acceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art as exemplified by Remington's Pharmaceutical Sciences, 16thEd. Mack Publishing Company, 1980, incorporated herein by reference. Itshould be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

The therapeutically effective doses are readily determinable using ananimal model, as shown in the studies detailed herein (see, e.g.,Example III). Experimental animals bearing solid tumors are frequentlyused to optimize appropriate therapeutic doses prior to translating to aclinical environment. Such models are known to be very reliable inpredicting effective anti-cancer strategies. For example, mice bearingsolid tumors, such as used in Example III, are widely used inpre-clinical testing.

The inventors have used mice with C1300 (Mo8) tumors to determinetoxicity limits and working ranges of bispecific that give optimalanti-tumor effects with minimal toxicity.

It is currently proposed that effective doses for use in the treatmentof cancer will be between about 0.1 mg/kg and about 2 mg/kg, andpreferably, of between about 0.8 mg/kg and about 1.2 mg/kg, whenadministered via the IV route at a frequency of about 1 time per week.Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Such optimization and adjustment is routinelycarried out in the art and by no means reflects an undue amount ofexperimentation.

It should be remembered that one aspect of the present inventionconcerns the delivery of a coagulating agent to a tumor site byadministering an uncomplexed bispecific binding ligand that garners anendogenous coagulation factor from the circulation and concentrates itwithin the tumor site. In these cases, the pharmaceutical compositionsemployed will contain a ligand having a targeting and coagulant bindingregion, but will otherwise generally be the same as those describedabove.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms are also contemplated, e.g., tablets or other solidsfor oral administration, time release capsules, liposomal forms and thelike. Other pharmaceutical formulations may also be used, dependent onthe condition to be treated. For example, topical formulations that areappropriate for treating pathological conditions such as dermatitis andpsoriasis; and ophthalmic formulations for diabetic retinopathy.

2. Ingestible Formulations

In certain embodiments, active compounds may be administered orally.This is contemplated for agents that are generally resistant, or havebeen rendered resistant, to proteolysis by digestive enzymes. Suchcompounds are contemplated to include chemically designed or modifiedagents; dextrorotatory peptidyl agents; liposomal formulations; andformulations in time release capsules to avoid peptidase and lipasedegradation.

For oral administration, the active bispecific compounds may beadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or compressed into tablets, or incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active bispecificcoagulant. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 60%of the weight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier.

Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, or capsules may be coated with shellac, sugar or both. A syrup ofelixir may contain the active compounds sucrose as a sweetening agentmethyl and propylparabens as preservatives, a dye and flavoring, such ascherry or orange flavor. of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compounds maybe incorporated into sustained-release preparation and formulations.

3. Liposomal Formulations

The bispecific coagulating ligands of the present invention may also beformulated in liposomal preparations if desired. The followinginformation may be utilized in generating liposomal formulationsincorporating the present coagulants. Phospholipids form liposomes whendispersed in water, depending on the molar ratio of lipid to water. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins such as cytochrome cbind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for use with the presentinvention will contain cholesterol, or even PEG.

The ability to trap solutes varies between different types of liposomes.For example, multilamellar vesicles (MLVs) are moderately efficient attrapping solutes, but small unilamellar vesicles (SUVs) are inefficient.SUWs offer the advantage of homogeneity and reproducibility in sizedistribution, however, and a compromise between size and trappingefficiency is offered by large unilamellar vesicles (LUVs). These areprepared by ether evaporation and are three to four times more efficientat solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity and surface charge.They may persist in tissues for hours or days, depending on theircomposition, and half lives in the blood range from minutes to severalhours. Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior dictates that liposomes concentrate only in those organs andtissues accessible to their large size. As this clearly includes theblood, this is not a limitation to their combined use with the presentinvention.

In other embodiments, the bispecific components of the invention may beadmixed with the liposome surface to direct the drug contents to thespecific antigenic receptors located on the target cell surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

4. Topical Formulations

The formulation of bispecific coagulants for topical use, such as increams, ointments and gels is also contemplated. The preparation ofoleaginous or water-soluble ointment bases is also well known to thosein the art. For example, these compositions may include vegetable oils,animal fats, and more preferably, semisolid hydrocarbons obtained frompetroleum. Particular components used may include white ointment, yellowointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum,white petrolatum, spermaceti, starch glycerite, white wax, yellow wax,lanolin, anhydrous lanolin and glyceryl monostearate.

Various water-soluble ointment bases may also be used, including glycolethers and derivatives, polyethylene glycols, polyoxyl 40 stearate andpolysorbates. Even delivery through the skin may be employed if desired,e.g., by using transdermal patches, iontophoresis or electrotransport.

5. Ophthalmic Formulations

The bispecific coagulating ligands of the present invention may also beformulated into pharmaceutical compositions suitable for use asophthalmic solutions. Such ophthalmic solutions are of interest, forexample, in the treatment of diabetic retinopathy. Thus, for thetreatment of diabetic retinopathy a bispecific conjugate of thisinvention would be administered to the eye of the subject in need oftreatment in the form of an ophthalmic preparation prepared inaccordance with conventional pharmaceutical practice, see for example"Remington's Pharmaceutical Sciences" 15th Edition, pages 1488 to 1501(Mack Publishing Co., Easton, Pa.).

The ophthalmic preparation will contain a novel bispecific coagulant ora pharmaceutically acceptable salt thereof in a concentration from about0.01 to about 1% by weight, preferably from about 0.05 to about 0.5% ina pharmaceutically acceptable solution, suspension or ointment. Somevariation in concentration will necessarily occur, depending on theparticular compound employed, the condition of the subject to be treatedand the like, and the person responsible for treatment will determinethe most suitable concentration for the individual subject. Theophthalmic preparation will preferably be in the form of a sterileaqueous solution containing, if desired, additional ingredients, forexample preservatives, buffers, tonicity agents, antioxidants andstabilizers, nonionic wetting or clarifying agents, viscosity-increasingagents and the like.

Suitable preservatives for use in such a solution include benzalkoniumchloride, benzethonium chloride, chlorobutanol, thimerosal and the like.Suitable buffers include boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium carbonate, sodiumacetate, sodium biphosphate and the like, in amounts sufficient tomaintain the pH at between about pH 6 and pH 8, and preferably, betweenabout pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran70, dextrose, glycerin, potassium chloride, propylene glycol, sodiumchloride, and the like, such that the sodium chloride equivalent of theophthalmic solution is in the range 0.9 plus or minus 0.2%.

Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, thiourea and the like. Suitablewetting and clarifying agents include polysorbate 80, polysorbate 20,poloxamer 282 and tyloxapol. Suitable viscosity-increasing agentsinclude dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like. Theophthalmic preparation will be administered topically to the eye of thesubject in need of treatment by conventional methods, for example in theform of drops or by bathing the eye in the ophthalmic solution.

6. Therapeutic Kits

The present invention also provides therapeutic kits comprising thebispecific coagulating ligands described herein. Such kits willgenerally contain, in suitable container means, a pharmaceuticallyacceptable formulation of at least one bispecific ligand in accordancewith the invention. The kits may also contain other pharmaceuticallyacceptable formulations, such as those containing additional bispecificcoagulating ligands, generally those having a distinct targetingcomponent; extra uncomplexed coagulation factors; bispecific antibodies,T cells, or other functional components for use in, e.g., antigeninduction; components for use in antigen suppression, such as acyclosporin; distinct anti-tumor site antibodies or immuntoxins; and anyone or more of a range of chemotherapeutic drugs.

Preferred agents for use in combination kits are inducing agents capableof inducing disease-associated vascular endothelial cells to express atargetable antigen, such as E-selectin or an MHC Class II antigen.Inducing agents can include T cell clones that bind disease or tumorantigens and that produce IFN-γ. Preferred inducing agents includebispecific antibodies that bind to disease or tumor cell antigens and toeffector cells capable of inducing target antigen expression through theelaboration of cytokines.

As such, the present invention further includes kits that comprise, insuitable container means, a first pharmaceutical composition comprisinga bispecific antibody that binds to an activating antigen on an effectorcell surface, i.e., a monocyte/macrophage, mast cell, T cell or NK cell,and to an antigen on the cell surface of disease cell; and a secondpharmaceutical composition comprising a bispecific ligand that comprisesa first binding region that binds to an endothelial cell antigen inducedby an activated effector cell, or cytokine therefrom, where the firstbinding region is operatively linked to a coagulation factor or a secondbinding region that binds to a coagulation factor.

Kits including a first pharmaceutical composition that comprises abispecific antibody that binds to the activating antigen CD14, CD16 (FcRfor IgE), CD2, CD3, CD28 or the T-cell receptor antigen are preferred,with CD14 or CD28 binding bispecific antibodies being more preferred.Activation of monocyte/macrophages or mast cells via CD14 or CD16binding results in IL-1 production that induces E-selectin; whereasactivation of T cells via CD2, CD3 or CD28 binding results in IFN-γproduction that induces MHC class II. Kits that include a secondpharmaceutical composition that comprises a bispecific ligand thatcomprises a first binding region that binds to E-selectin or to an MHCClass II antigen are therefore also preferred.

The kits may have a single container means that contains the bispecificcoagulating ligand, with or without any additional components, or theymay have distinct container means for each desired agent. Kitscomprising the separate components necessary to make a bispecificcoagulating ligand are also contemplated.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. However, the componentsof the kit may be provided as dried powder(s). When reagents orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

The container means of the kit will generally include at least one vial,test tube, flask, bottle, syringe or other container means, into whichthe bispecific coagulating ligand, and any other desired agent, may beplaced and, preferably, suitably aliquoted. Where additional componentsare included, the kit will also generally contain a second vial or othercontainer into which these are placed, enabling the administration ofseparated designed doses. The kits may also comprise a second/thirdcontainer means for containing a sterile, pharmaceutically acceptablebuffer or other diluent.

The kits may also contain a means by which to administer the bispecificligand to an animal or patient, e.g., one or more needles or syringes,or even an eye dropper, pipette, or other such like apparatus, fromwhich the formulation may be injected into the animal or applied to adiseased area of the body. The kits of the present invention will alsotypically include a means for containing the vials, or such like, andother component, in close confinement for commercial sale, such as,e.g., injection or blow-molded plastic containers into which the desiredvials and other apparatus are placed and retained.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I Synthesis of a Bispecific Coagulating Antibody

The present example describes the synthesis of a bispecific antibodycapable of specifically directing a coagulant to a tumor site, i.e., a"coaguligand".

A. Materials and Methods

1. Reagents

Pepsin (A; EC 3.4.23.1), Ellmans reagent (ER;5,5'-dithio-bis(2-nitrobenzoic acid, DNTB), 2-mercaptoethanol (2-ME),sodium arsenite (NaAsO₂) and rabbit brain thromboplastin (acetonepowder) were obtained from Sigma Chemical Co., St. Louis Mo. SephadexG-25 and G-100 were obtained from Pharmacia LKB (Piscataway, N.J.).

2. Human Truncated Tissue Factor (tTF)

Recombinant human truncated TF (tTF) was prepared by one of twodifferent methods.

Method I: Construction of the E. coli Expression Vector. The cDNA codingfor tTF (residues 1-218) was amplified by PCR using the primers5'-GAAGAAGGGATCCTGGTGCCTCGTGGTTCTGGCACTACAAATACT-3' (5'-primer; SEQ IDNO:28) and 5'-CTGGCCTCAAGCTTAACGGAATTCACCTTT-3' (3'-primer; SEQ IDNO:29) which allowed the addition of the coding sequence for a thrombincleavage site upstream of the cDNA. The PCR products were cleaved usingBamHI and HindIII and ligated between the BamHI and HindIII sites of theexpression vector pTrcHisC (Invitrogen).

DH5α cells were transformed with the ligation mixture and recombinantplasmids were isolated after selection in the presence of ampicillin.The E. coli strain BL21 was transformed with the recombinant plasmidpTrcHisC-tTF and the resultant transformants were used for proteinexpression.

Method I: Expression, Refolding and Purification of tTF from E. coli.The poly(his)-tTF fusion protein was expressed using BL21 cellstransformed with pTrc-HisC-tTF. Inoculant cultures (10 ml in LB medium)were grown overnight shaking at 37° C.

Inoculant cultures were added to growth medium which were then grownshaking at 37° C. When the optical density at 550 nm had reach ca. 0.5,10 ml of 100 mM isopropyl-β-D-thiogalactopyranoside was added. Shakingwas continued at 37° C. for ca. 20 h (to stationary phase).

The cells were harvested by centrifugation (10,000×g, 20 min.) and theinclusion bodies were isolated as follows (quantities of reagents areper gram of cell paste). The cell paste was suspended in 4 ml of 10 mMTris, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 0.17 mg/ml PMSF, 2 mg/ml hen eggwhite lysozyme (Sigma). Benzonase (250 units, EM Science) was added thesuspension was mixed gently at room temperature for 1.5 h thencentrifuged at 12,000 g for 15 min.

The pellet was resuspended in 10 mM Tris, pH 7.5, 1 mM EDTA, 3% NP40 (2ml), sonicated for 1 min at 50% power and centrifuged at 12,000×g for 20min. The pellet was resuspended in water, sonicated for 20-30 seconds at50% power and centrifuged at 12,000×g for 20 min. The water wash wasrepeated and the final pellet, highly enriched for the inclusion bodies,was suspended in 6 M guanidinium chloride, 0.5 M NaCl, 20 mM phosphate,10 mM β-mercaptoethanol, pH 8.0 (9 ml per gram of inclusion bodies) bygentle mixing at room temperature overnight.

The suspension was centrifuged at 12,000×g for 20 min and thesupernatant was loaded onto a nickel nitriloacetic acid (Ni-NTA, Qiagen)column. The column was washed successively with the same 6 M guanidiniumchloride buffer at pH 8 then pH 7, then the protein was eluted bydecreasing the pH to 4.

Ni-NTA column fractions containing the fusion protein were combined anddithiothreitol was added to 50 mM. The solution was held at roomtemperature overnight then diluted to a protein concentration of ca. 1mg/ml in 6 M urea, 50 mM Tris, 0.02% sodium azide, pH 8.0 and dialyzedat 4° C. overnight against 10-20 volumes of the same buffer. The bufferwas changed to 2 M urea, 50 mM Tris, 300 mM NaCl, 2.5 mM reducedglutathione, 0.5 mM oxidized glutathione, 0.02% sodium azide, pH 8.0(folding buffer). Dialysis was continued for 2 more days, the buffer wasreplaced by fresh folding buffer and dialysis was continued for 2 moredays.

The solution was then dialyzed extensively against 20 mM TEA (pH 7.5),removed from the dialysis bag, treated with human thrombin (ca. 1 partper 500 parts recombinant protein w/w) overnight at room temperature,and loaded onto a HR-10/10 mono-Q anion exchange column. tTF protein waseluted using a 20 mM TEA buffer containing NACl in a concentrationincreasing linearly from 0 to 150 mM over 30 minutes (flow rate 3ml/min).

Method II: Preparation of tTF complimentary DNA (cDNA). RNA from J-82cells (human bladder carcinoma) was used for the cloning of tTF. TotalRNA was isolated using the GlassMax™ RNA microisolation reagent (GibcoBRL). The RNA was reverse transcribed to cDNA using the GeneAmp RNA PCRkit (Perkin Elmer). tTF cDNA was amplified using the same kit with thefollowing two primers:

    5' primer:     5' GTC ATG CCA TGG CCC TGG TGC CTC GTG CTT                                                         (SEQ ID NO:2)                                   CTG GCA CTA CAA ATA CT                                                  3' primer:  5' TGA CAA GCT TAT TCT CTG AAT TCC CCC TTT CT                 

The underlined sequences codes for the N-terminus and C-terminus of tTF.The rest of the sequence in the 5' primer is the restriction site forNcoI allowing the cloning of tTF into the expression vector and codesfor a cleavage site for thrombin. The sequence in the 3' primer is theHindIII site for cloning tTF into the expression vector. PCRamplification was performed as suggested by the manufacturer. Briefly,75 μM dNTP; 0.6 μM primer, 1.5 mM MgCl₂ were used and 30 cycles of 30"at 95° C., 30" at 55° C. and 30" at 72° C. were performed.

Method II. Vector Constructs. E. coli expression vector H₆ pQE-60 wasused for expressing tTF (Lee et. al., 1994). The PCR amplified tTF cDNAwas inserted between the NcoI and HindIII site. H₆ pQE-60 has a built-in(His)₆ encoding sequence such that the expressed protein has thesequence of (His)₆ at the N-terminus, which can be purified on theNi-NTA column.

Method II. tTF Purification. tTF containing H₆ pQE-60 DNA wastransformed to E. coli TG-1 cells. The cells were grown to OD₆₀₀ =0.5and IPTG was added to 30 μM to induce the tTF production. The cells wereharvested after shaking for 18 h at 30° C. The cell pellet was denaturedin 6 M Gu-HCl and the lysate was loaded onto a Ni-NTA column (Qiagen).The bound tTF was washed with 6 M urea and tTF was refolded with agradient of 6 M-1 M urea at room temperature for 16 h. The column waswashed with wash buffer (0.05 Na H₂ PO₄, 0.3 M NaCl, 10% glycerol) andtTF was eluted with 0.2 M Imidozole in wash buffer.

The eluted tTF was concentrated and loaded onto a G-75 column. tTFmonomers were collected and treated with thrombin to remove the H₆peptide. This was done by adding 1 part of thrombin (Sigma) to 500 partsof tTF (w/w), and the cleavage was carried out at room temperature for18 h. Thrombin was removed from tTF by passage of the mixture through aBenzamidine Sepharose 6B thrombin affinity column (Pharmacia).

The tTF had identical ability to recombinant tTF from yeast or Chinesehamster ovary cells to bind factor VIIa and to enhance the catalyticactivity of VIIa (Ruf et al., 1991). When analyzed by polyacrylamide gelelectrophoresis in sodium dodecyl sulfate, it ran as a single componenthaving a molecular weight of approximately 24 kD.

3. Monoclonal Antibodies

B21-2 (TIB-229) hybridoma and SFR8-B6 hybridoma (HB-152, hereafterreferred to as SFR8) were obtained from the ATCC. Both hybridomassecreted rat IgG2b antibodies, which were purified from culturesupernatant by protein G affinity chromatography. The B21-2 antibodyreacts with I-A^(d) antigen expressed on A20 cells as well as on thevasculature of the C1300 (Muγ) transfectant tumors grown in BALB/c/nu/numice. SFR8 antibody is directed against the HLA-Bw6 epitope and servesas an isotype matched negative control for the B21-2 antibody.

TF9/10H10 (referred to as 10H10), a mouse IgG1, is reactive with humanTF without interference of TF/factor VIIa activity and was produced asdescribed by Morrissey et al. (1988).

The cell line MRC OX7 (referred to as OX7) was obtained from Dr. A. F.Williams (MRC Cellular Immunology Unit, University of Oxford, Oxford,England). It secretes the OX7 antibody, a mouse IgG1 antibody thatrecognizes the Thy 1.1 antigen on T lymphocytes. It was used as anisotype matched negative control for TF9/10H10.

All antibodies were purified from culture supernatant by protein Gaffinity chromatography.

4. Synthesis of Bispecific Antibodies

F(ab')₂ fragments were obtained by digesting their respective IgGs with2% (w/v) pepsin for 5-9 hrs at 37° C. and purification of the fragmentsby Sephadex G100 chromatography. Synthesis of the bispecific antibodiesB21-2/10H10, SFR8/10H10 and B21-2/OX7 was carried out according to themethod of Brennan et al. (1985) with minor modifications.

The bispecific antibodies B21-2/10H10, SFRB/10H10, OX7/10H10 andB21-2/OX7 were synthesized according to the method of Brennan et al.(1985) with minor modifications. In brief, F(ab')₂ fragments wereobtained from the IgG antibodies by digestion with pepsin and werepurified to homogeneity by chromatography Sephadex G100. F(ab')₂fragments were reduced for 16 h at 20° C. with 5 mM 2-mercaptoethanol in0.1 M sodium phosphate buffer, pH 6.8, containing 1 mM EDTA (PBSEbuffer) and 9 mM NaAsO₂. Ellman's reagent (ER) was added to give a finalconcentration of 25 mM and, after 3 h at 20° C., theEllman's-derivatized Fab' fragments (Fab'-ER) were separated fromunreacted ER on columns of Sephadex G25 in PBSE.

To form the bispecific antibody, Fab'-ER derived from one antibody wasconcentrated to approximately 2.5 mg/ml in an ultrafiltration cell andwas reduced with 10 mM 2-mercaptoethanol for 1 h at 20° C. The resultingFab'-SH was filtered through a column of Sephadex G25 in PBSE and wasmixed with equal molar quantities of Fab'-ER prepared from the secondantibody. The mixtures were concentrated by ultrafiltration toapproximately 3 mg/ml and were stirred for 16 h at 20° C. The productsof the reaction were fractionated on columns of Sephadex G100 and thefractions containing the bispecific antibody (110 kDa) were concentratedto 1 mg/ml, and were stored at 4° C. in 0.02% sodium azide.

B. Results

1. Analysis of Bispecific Antibodies

The molecular weight of the F(ab')₂ fragments and bispecificpreparations were determined by SDS-Page electrophoresis with 4-15%gradient gels using the Pharmacia LKB-Phastsystem (Pharmacia LKB,Piscataway, N.J.). Bispecificity as well as the percentage ofheterodimer vs homodimer was determined by FACS analysis (Example II).

Analysis of the bispecific antibodies by SDS-Page electrophoresis (andby FACS, Example II) demonstrated that the B21-2/10H10 bispecificcontained less than 4% homodimer of either origin and <10% fragmentswith a molecular weight of 140 kD or 55 kD. Approximately 10% of thepreparation consisted of 140 kD fragments, probably being a F(ab')₂construct with an extra light chain (of either origin) attached.

EXAMPLE II Coagulating Antibody Binding and Function In Vitro

The present example shows the bispecificity of the coagulating antibody(coaguligand) and demonstrates that specific binding, cellular deliveryand coagulation is achieved in vitro using the coaguligand.

A. Materials and Methods

1. Cells

The A20 cell line, which is an I-A_(d) positive BALB/c B-cell lymphoma,was purchased from the American Type Culture Collection (ATCC;Rockville, Md; TIB-208). A20 cells were grown in DMEM supplemented with10% (v/v) fetal calf serum (FCS), 0.2 mM L-glutamine, 200 units/mlpenicillin and 100 μg/ml streptomycin, 18 mM Hepes, 0.1 mM non-essentialamino acids mix and 1 mM sodium pyruvate (medium hereafter referred toas complete DMEM; all reagents obtained from Life Technologies,Gaitherburg, Md.). 2-ME is added to complete DMEM to a finalconcentration of 0.064 mM for A20 cells. Cultures were maintained at 37°C. in a humidified atmosphere of 90% air/10% CO₂.

J82, a human gall bladder carcinoma expressing TF, was obtained from theATCC (HTB-1). The cells grew adherently in complete DMEM.

The C1300 neuroblastoma cell line was established from a spontaneoustumor, which arose in an A/Jax mouse (Dunham & Stewart, 1953). The C1300(Muγ) 12 line, hereafter referred to as C1300 (Muγ) was derived bytransfection of C1300 neuroblastoma cells with the murine IFN-γ geneusing the IFN-γ expression retrovirus PSVX (Muγ ΔAs) (Watanabe et al.,1989). The IFN-γ expression retrovirus was obtained from Dr. Y. Watanabe(Department of Molecular Microbiology, Kyoto University, Japan).

C1300(Muγ)12 cells were maintained in Dulbecco's modified Eagle Medium(DMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 2.4 mML-glutamine, 200 units/ml penicillin, 100 μg/ml streptomycin, 100 μMnonessential amino acids, 1 μM sodium pyruvate, 18μM HEPES and 1 mg/mlG418 (Geneticin; Sigma). Cultures were maintained at 37° C. in ahumidified atmosphere of 90% air/10% CO₂.

The Thy 1.1-expressing AKR-A mouse T lymphoma cell line was obtainedfrom Prof. Dr. I. MacLennan (Department of Experimental Pathology,Birmingham University, Birmingham, England) and were grown in completeDMEM.

2. Indirect Immunofluorescence

A20 cells were resuspended in PBS/0.2% BSA/0.02% Na-azide (hereafterreferred to as FACS buffer) at 4×10⁶ cells/ml. J82 cells were releasedfrom the flask under mild conditions using PBS/EDTA (0.2% w/v) andresuspended at 4×10⁶ cells/ml in FACS buffer. 50 μl of cell suspensionwas added to 50 μl of optimal serial dilutions of the primary antibodyin wells of a round-bottomed 96 well plate. After incubation at RT for15 min, the cells were washed with FACS buffer 3 times. After removingthe final supernatant, 50 μl of the secondary antibody conjugated tofluorescein isothiocyanate (FITC), in a 1 in 20 dilution in FACS buffer,was added to the cells. The cells were incubated for a further 15 min atRT and washed 3 times with FACS buffer. Cell associated fluorescence wasmeasured on a FACScan (Becton Dickenson, Fullerton, Calif.). Data wereanalyzed using the Lysis II program. When FITC-anti-rat immunoglobulinwas used as the secondary antibody, normal mouse serum (10% v/v) wasadded to block non-specific cross reactivity with the mouse cells.

3. Radiolabeling of Proteins

Proteins were labeled with ¹²⁵ Iodine according to the chloramine Tprotocol described by Mason & Williams (1980), (protocol 2). Theiodinated product was purified on G25 and stored at -70° C. in thepresence of 5% DMSO and 5 mg/ml bovine IgG in the case of the monoclonalfragments and 5% DMSO and 5 mg/ml BSA in the case of tTF. Specificactivity ranged between 2.5 μCi/μg and 4.8 μCi/μg.

4. Binding Studies

Human tTF was labelled with ¹²⁵ I to a specific activity of 2.5-4.8μCi/μg using the chloramine T procedure (Protocol 2) described by Masonand Williams (1980). A suspension of A20 cells at 2×10⁶ cells/ml in PBScontaining 2 mg/ml BSA and 0.02% sodium azide was distributed in 50 μlvolumes into the wells of 96 well round-bottomed microtiter plates. Tothe wells were added 25 μl of bispecific antibodies prepared over arange of concentrations (8 to 0.02 μg/ml) in the same buffer.

25 μl of ¹²⁵ I-tTF at 8 μg/ml in the same buffer were added to eachwell, giving a molar excess of tTF. The plates were shaken and incubatedfor 1 hr at 4° C. The cells were then washed 3× in the plates with 0.9%(w/v) NaCl containing 2 mg/ml BSA. The contents of the wells werepipetted over a 10:11 (v/v) mixture of dibutyl phthalate andbis(2-ethylhexyl)phthalate oils in microcentrifuge tubes. The tubes werecentrifuged for 1.5 min at 7500 g and were snap frozen in liquidnitrogen. The tips containing the cells were cut off. The radioactivityin the cell pellet and in the supernatant was measured in a gammacounter.

5. Coaculation Assay

An identical microplate to that used for the binding assay above was setup on the same occasion, except that non-labelled tTF was added insteadof ¹²⁵ I-tTF. After the 1h incubation at 4° C., the cells were washed 3×as before and were resuspended in 75 μl of 0.9% NaCl containing 2 mg/mlBSA and 12.5 mM CaCl₂. The contents of the wells were transferred to 5ml clear plastic tubes and were warmed to 37° C. To each tube was added30 μl of citrated mouse plasma at 37° C. The time for the first fibrinstrands to form was recorded.

B. Results

1. Antibody Bispecificity

For SFR8/10H10 bispecificity was shown by FACS using J82 cells (TFpositive) as target cells and FITC-anti-mouse immunoglobulin todemonstrate 10H10 presence. FITC-anti-rat immunoglobulin was used todemonstrate the presence of SFR8. The mean fluorescenceintensity-versus-concentration curves were coincident for both stains,demonstrating that both the mouse and the rat arm are present in thebispecific preparation.

2. Antibody Binding

Binding studies with ¹²⁵ Iodine labeled B21-2 Fab' and SFR8 Fab' showedthat the concentration at which saturation of binding of B21-2 Fab' toA20 cells is reached is 21.5 nM. The SFR8 Fab' bound non-specifically toA20 cells, with the number of molecules bound per cell being less than50,000 at 21.5 nM versus 530,000 for B21-2 Fab'.

3. Coagulant Delivery and Tethering

To study the capability of bridging tTF to A20 cells through theB21-2/10H10 bispecific antibody as compared to the control bispecificantibodies, A20 cells were incubated with bispecific antibody and a ¹²⁵I-tTF concentration range as indicated. Saturation was attained atconcentrations of bispecific antibody of 10 nM (1 μg/ml) or more, whenan average of 310,000 molecules of tTF were bound to each A20 cell. Thebinding was specific since no tTF binding was mediated by either of theisotype-matched control bispecific antibodies, SFR8/10H10 or B21-2/OX7,which had only one of the two specificities needed for tethering tTF(FIG. 1).

4. Coagulation

To investigate whether tTF bound to A20 cells through a bispecificantibody was able to induce coagulation, the inventors first incubatedA20 cells with 21.5 nM bispecific antibody and 69 nM tTF. The resultingeffect on the coagulation time is shown in Table VII. These firststudies showed that A20 cells coated with a complex of B21-2/10H10 andtTF were capable of inducing fibrin formation: it shortened coagulationtime from 140 sec (the time for mouse plasma in CaCl₂ to coagulate inthe absence of added antibodies or TF under the specific conditionsused) to 60 sec. In contrast, the control bispecific antibodies did notinduce activation of coagulation: in these cases coagulation time was140 sec.

Later studies confirmed and extended the initial results. Mouse plasmaadded to A20 cells to which tTF had been tethered with B21-2/10H10coagulated rapidly. Fibrin strands were visible 36 seconds after addingthe plasma as compared with 164 seconds in plasma added to untreated A20cells (Table VII). Only when tTF had been tethered to the cells wascoagulation induced: no effect on coagulation time was seen with cellsincubated with of tTF alone, homodimeric F(ab')₂, Fab' fragments orbispecific antibodies having only one of the two specificities neededfor tethering tTF.

A linear relationship existed between the logarithm of the averagenumber of tTF molecules tethered to each A20 cell and the rapidity withwhich those cells induced coagulation of mouse plasama (FIG. 2). Cellsbearing 300,000 molecules of tTF per cell induced coagulation in 40 secsbut even with 20,000 molecules per cell coagulation was significantlyfaster (140 secs) than it was with untreated cells (190 secs).

                  TABLE VII                                                       ______________________________________                                        Coagulation of mouse plasma induced by tethering                              tTF to A20 cells with bispecific antibody.                                    Reagents added.sup.1                                                                         Coagulation time.sup.2 (sec)                                   ______________________________________                                        None           164 ± 4                                                     B21-2/10H10 + tTF                                                                                36 ± 2                                                  B21-2/10H10           163 ± 2                                              tTF                     163 ± 3                                            B21-2/OX7 + tTF                                                                                    165 ± 4                                               SFR8/10H10 + tTF                                                                                     154 ± 5                                             10H10 F(ab').sub.2 + tTF                                                                          160 ± 3                                                10H10 Fab' + tTF                                                                                    162 ± 2                                              B21-2 F(ab').sub.2 + tTF                                                                          168 ± 4                                                B21-2 Fab' + tTF                                                                                    165 ± 4                                              ______________________________________                                         .sup.1 Bispecific antibodies F(ab').sub.2 and Fab' fragments (0.33            μg/10.sup.5 cells/100 μl) and tTF (0.17 μg/10.sup.5 cells/100        μl) were incubated with A20 cells for 1 h at 4° C. in 0.2% w/v      sodium azide. The cells were washed, warmed to 37° C., calcium and     plasma were added and the time for the first fibrin strands to form was       recorded.                                                                     .sup.2 Arithmetic mean of triplicate determinations ± standard             deviation                                                                

EXAMPLE III Specific Tumor Vasculature Specific Coagulation In Vivo

The present example describes the specific coagulation of tumorvasculature in vivo that results following the administration of thebispecific antibody coaguligand as a delivery vehicle for human tissuefactor.

A. Materials and Methods

1. Reagents

Mouse blood was obtained by heartpuncture and collected in 1/10 volumeof 3.8% buffered citrate. The blood was centrifuged for 10 min at 3000 gand the plasma snap frozen in small aliquots and stored at -70° C.

2. Animals

BALB/c nu/nu mice were obtained from Simonsen (Gilroy, Calif.) andmaintained under SPF conditions.

3. C1300 (Muγ) Mouse Model and Treatment

The tumor model was as previously described (Burrows et al., 1992;Burrows & Thorpe, 1993) with three refinements. First, a differentantibody, B21-2, was used. This antibody recognizes I-A^(d) but notI-E^(d), unlike the previously used M5/114 antibody which recognizesboth molecules. The B21-2 antibody has an approximately 10-fold betteraffinity than M5/114. Second, a subline of the previously usedC1300(Muγ)12 line was used which grew continuously in BALB/c nu/nu mice.The C1300(Muγ) 12 cells used previously had to be mixed withuntransfected C1300 cells in order to form continuously growing tumors.The new subline, designated C1300(Muγ) t1P3, will be referred tohereafter as C1300(Muγ). Third, it was unnecessary to add tetracyclineto the mice's drinking water to prevent gut bacteria from inducingI-A^(d) on the gastrointestinal epithelium. Unlike immunotoxins,coaguligands do not damage I-A^(d) -expressing intestinal epithelium.

For establishment of solid tumors, 1.5×10⁷ C1300 (Muγ) cells wereinjected subcutaneously into the right anterior flank of BALB/c nu/numice. When the tumors had grown to 0.8 cm in diameter, mice wererandomly assigned to different treatment groups each containing 7-8mice.

Coaguligands were prepared by mixing bispecific antibodies (140 μg) andtTF (110 μg) in a total volume of 2.5 ml of 0.9% NaCl and leaving themat 4° C. for one hour. Mice then received intravenous injections of 0.25ml of this mixture (i.e. 14 μg of bispecific antibody plus 11 μg oftTF). Other mice received 14 μg of bispecific antibodies or 11 μg of tTFalone. The injections were performed slowly into one of the tail veinsover approximately 45 sec and were followed with a second injection of200 μl of saline into the same vein. This injection procedure wasadopted to prevent thrombosis of the tail vein which was seen if micewere rapidly injected (1-2 sec). Seven days later, the treatments wererepeated.

Perpendicular tumor diameters were measured at regular intervals andtumor volumes were estimated according to the following equation:

    volume=smaller diameter.sup.2 ×larger diameter×π/6

Differences in tumor volume were tested for statistical significanceusing the Mann-Whitney-Wilcoxon nonparametric test for two independentsamples (Gibbons, 1976).

For histopathological analyses, animals were anesthetized with metophaneat various times after treatment and were exsanguinated by perfusionwith heparinized saline. 500 IU of heparin were i.v. injected, theanimal anesthetized with metofane and the systemic circulation perfusedwith PBS at a flow rate of 0.6 mls/min until the liver had been clearedof blood. The tumor and normal tissues were excised and formalin fixed(4% v/v). Paraffin sections of the tissues were cut and stained with thestandard Martius Scarlet Blue (MSB) trichrome technique for detection offibrin, and with hematoxylin and eosin stain for cell morphology.

B. Results

1. Improved Tumor Model

To improve the C1300 (Muγ) tumor model as described before (Burrows etal., 1992), the inventors subcloned the C1300 (Muγ) cell line into acell line that can grow without being mixed with its parental cell,C1300, but still express the I-A^(d) MHC Class II antigen on theendothelial cells of the tumor. The inventors used an anti-I-A^(d)antibody (B21-2) that has a 5-10 fold higher affinity for its antigenthan the initial anti-I-A^(d) antibody (M5/114.15.2) used in this modelas determined by FACS. In vivo distribution studies with this newanti-I-A^(d) antibody showed the same tissue distribution pattern as didM5/114.15.2. Intense staining with B21-2 was seen in tumor vascularendothelium, light to moderate staining in Kuppfer cells in the liver,the marginal zones in the spleen and some areas in the small and largeintestines. Vessels in other normal tissues were unstained.

2. Determination of Suitable In Vivo Doses

The maximum tolerated dose was 16 μg B21-2/10H10 plus 11 μg tTF injectedintravenously into the tail vein of mice. At this dose, mice lost noweight and had normal appearance and activity levels. At a higher doseof 20 μg B21-2/10H10 plus 16 μg tTF, two of ten mice developed localizeddermal hemorrhages which eventually resolved. The lower dose was adoptedfor in vivo studies. Truncated TF itself was not toxic at 50 μg, givenintravenously.

3. Specific Coagulation and Infarction in Tumor Vasculature

Intravenous administration of a coaguligand composed of B21-2/10H10 (20μg) and tTF (16 μg) to mice bearing solid C1300 (Muγ) tumors causedtumors to assume a blackened, bruised appearance within 30 minutes. Ahistological study of the time course of events within the tumorrevealed that 30 minutes after injection of coaguligand all vessels inall regions of the tumor were thrombosed (FIG. 3B). Vessels containedplatelet aggregates, packed red cells and fibrin. At this time, tumorcells were healthy, being indistinguishable morphologically from tumorcells in untreated mice (FIG. 3A).

By 4 hours, signs of tumor cell distress were evident. The majority oftumor cells had begun to separate from one another and had developedpyknotic nuclei (FIG. 3C). Erythrocytes were commonly observed in thetumor interstitium. By 24 hours, advanced tumor necrosis was visiblethroughout the tumor (FIG. 3D). By 72 hours, the entire central regionof the tumor had compacted into morphologically indistinct debris.

In one of three of the tumors examined, a viable rim of tumor cells 5-10cell layers thick was visible on the outskirts of the tumor where it wasinfiltrating into surrounding normal tissues. Immunohistochemicalexamination of serial sections of the same tumor revealed that thevessels in the regions of tumor infiltration lacked class II antigens.

Tumors from control mice which had received B21-2/10H10 bispecificantibody (20 μg) alone 30 minutes or 24 hours earlier showed no signs ofinfarction. Tumors from mice which received tTF (16 μg), alone or incombination with B21-2/OX7 or SFR8/10H10, showed no signs of infarction30 min after injection but 24 hours after injection, occasional vessels(about 20% of vessels overall) in the tumor were infarcted. Theseappeared to be most prevalent in the core of the tumor.

No thrombi or morphological abnormalities were visible in paraffinsections of liver, kidney, lung, intestine, heart, brain, adrenals,pancreas and spleen taken from tumor-bearing mice 30 minutes, 4 hoursand 24 hours after administration of coaguligand or tTF.

4. Tumor Regressions of Solid Tumors

FIG. 4 shows the results of a representative anti-tumor study in which acoaguligand composed of B21-2/10H10 and tTF was administered to micewith 0.8 cm diameter tumors. The tumors regressed to approximately halftheir pretreatment size. Repeating the treatment on the 7th day causedthe tumors to regress further, usually completely. In 5/7 animals,complete regressions were obtained. Two of the mice subsequentlyrelapsed four and six months later. These anti-tumor effects arestatistically highly significant (P<0.001) when compared with all othergroups.

Tumors in mice treated with tTF alone or with tTF mixed with theisotype-matched control bispecific antibodies, SFR8/10H10 or B21-2/OX7,grew more slowly than those in groups receiving antibodies or diluentalone. These differences were statistically significant (P<0.05) on days12-14. Thus, part of the anti-tumor effects seen with the B21-2/10H10coaguligand are attributable to a slight non-specific action of tTFitself.

At the end of the study, two mice which had been treated with diluentalone and which had very large tumors of 2.0 cm³ and 2.7 cm³ (i.e.10-15% of their body weight) were given coaguligand therapy. Both hadcomplete remissions although their tumors later regrew at the originalsite of tumor growth.

C. Discussion

The present studies show that soluble human tTF, possessing practicallyno ability to induce coagulation, became a powerful thrombogen for tumorvasculature when targeted by means of a bispecific antibody to tumorendothelial cells. In vitro coagulation studies showed that therestoration of thrombotic activity of tTF is mediated through itscross-linking to antigens on the cell surface.

tTF binds factors VII and VIIa with high affinity and enhances thecatalytic activity of VIIa but does not induce coagulation of plasmabecause the tTF:VIIa complex has to be associated with a membranesurface for efficient activation of factors IX and X (Ruf et al., 1991;Krishnaswamy et al., 1992). Tethering of tTF:VIIa to the cell surface bymeans of a bispecific antibody restores its ability to inducecoagulation by bringing the tTF:VIIa into close proximity to themembrane: the membrane phospholipid provides the surface on which thecoagulation-initiation complexes with factors IX or X can assemble andefficiently produce intermediates in the clotting process.

Administration of a coaguligand directed against class II to mice havingtumors with class II-expressing vasculature caused rapid thrombosis ofblood vessels throughout the tumor. This was followed by infarction ofthe tumor and complete tumor regressions in a majority of animals. Inthose animals where complete regressions were not obtained, the tumorsgrew back from a surviving rim of tumor cells on the periphery of thetumor where it had infiltrated into the surrounding normal tissues. Thevessels at the growing edge of the tumor lacked class II antigens, thusexplaining the lack of thrombosis of these vessels by the coaguligand.It is likely that these surviving cells would have been killed bycoadministering a drug acting on the tumor cells themselves, as wasfound previously (Burrows & Thorpe, 1993).

The anti-tumor effects of the coaguligand were similar in magnitude tothose obtained in the same tumor model with an immunotoxin composed ofanti-class II antibody and deglycosylated ricin A-chain (Burrows &Thorpe, 1993). One difference between the two agents is their rapidityof action. The coaguligand induced thrombosis of tumor vessels in lessthan 30 minutes whereas the immunotoxin took 6 hours to achieve the sameeffect. The immunotoxin acts more slowly because thrombosis is secondaryto endothelial cell damage caused by the shutting down of proteinsyntheses.

A second and important difference between the immunotoxin and thecoaguligand is that they have different toxic side effects. Theimmunotoxin caused a lethal destruction of class II-expressinggastrointestinal epithelium unless antibiotics were given to suppressclass II induction by intestinal bacteria. The coaguligand caused nogastrointestinal damage, as expected because of the absence of clottingfactors outside of the blood, but caused coagulopathies in occasionalmice when administered at high dosage.

The findings described in this report demonstrate the therapeuticpotential of targeting human coagulation-inducing proteins to tumorvasculature. For clinical application, antibodies or other ligands areneeded that bind to molecules that are present on the surface ofvascular endothelial cells in solid tumors but absent from endothelialcells in normal tissues. Tumor endothelial markers could be induceddirectly by tumor-derived angiogenesis factors (Folkman, 1985) orcytokines (Burrows et al., 1991; Ruco et al., 1990), or could relate tothe rapid proliferation (Denekamp & Hobson, 1982) and migration(Folkman, 1985) of endothelial cells during neovascularization.

Several candidate antibodies have been described. The antibody TEC-11,against endoglin is a particular example that binds selectively to humantumor endothelial cells.

Other antibodies include FB5, against endosialin (Rettig et al., 1992),E-9, against an endoglin-like molecule (Wang et al., 1993), BC-1,against a fibronectin isoform (Carnemolla et al., 1989) and TP-1 andTP-3, against an osteosarcoma-related antigen (Bruland et al., 1988).CD34 has been reported to be upregulated on migrating endothelial cellsand on the abluminal processes of budding capillaries in tumors andfetal tissues (Schlingemann et al., 1990). The receptors for vascularendothelial cell growth factor (VEGF) become upregulated in tumor bloodvessels (Plate et al., 1993; Brown et al., 1993) probably in response tohypoxia (Thieme et al., 1995), and selectively concentrate VEGF in tumorvessels (Dvorak et al., 1991).

The induction of tumor infarction by targeting coagulation-inducingproteins to these and other tumor endothelial cell markers is proposedas a valuable new approach to the treatment of solid tumors. Thecoupling of human (or humanized) antibodies to human coagulationproteins to produce wholly human coaguligands is particularlycontemplated, thus permitting repeated courses of treatment to be givento combat both the primary tumor and its metastases.

EXAMPLE IV Synthesis of Truncated Tissue Factor (tTF) Constructs

tTF is herein designated as the extracellular domain of the maturetissue factor protein (amino acid 1-219 of the mature protein; SEQ IDNO:23). SEQ ID NO:23 is encoded by, e.g., SEQ ID NO:22.

A. H₆ [tTF]

H₆ Ala Met Ala[tTF]. The tTF complimentary DNA (cDNA) was prepared asfollows: RNA from J-82 cells (human bladder carcinoma) was used for thecloning of tTF. Total RNA was isolated using the GlassMax™ RNAmicroisolation reagent (Gibco BRL). The RNA was reverse transcribed tocDNA using the GeneAmp RNA PCR kit (Perkin Elmer). tTF cDNA wasamplified using the same kit with the following two primers:

5' primer: 5' GTC ATG CCA TGG CCT CAG GCA CTA CAA (SEQ ID NQ:1)

3' Primer: 5' TGA CAA GCT TAT TCT CTG AAT TCC CCC TTT CT (SEQ ID NO:2)

The underlined sequences codes for the N-terminus of tTF. The rest ofthe sequence in the 5' primer is the restriction site for NcoI allowingthe cloning of tTF into the expression vector. The sequence in the 3'primer is the HindIII site for cloning tTF into the expression vector.PCR amplification was performed as suggested by the manufacturer.Briefly, 75 μM dNTP; 0.6 μM primer, 1.5 mM MgCl₂ were used and 30 cyclesof 30" at 95° C., 30" at 55° C. and 30" at 72° C. were performed.

The E. coli expression vector H₆ pQE-60 was used for expressing tTF (Leeet al., 1994). The PCR amplified tTF cDNA was inserted between the NcoIand Hind3 site. H₆ pQE-60 has a built-in (His)₆ encoding sequence suchthat the expressed protein has the sequence of (His)₆ at the N terminus,which can be purified on a Ni-NTA column.

To purify tTF, tTF containing H₆ pQE-60 DNA was transformed to E. coliTG-1 cells. The cells were grown to OD₆₀₀ =0.5 and IPTG was added to 30μM to induce the tTF production. The cells were harvested after shakingfor 18 h at 30° C. The cell pellet was denatured in 6 M Gu-HCl and thelysate was loaded onto a Ni-NTA column (Qiagen). The bound tTF waswashed with 6 M urea and tTF was refolded with a gradient of 6 M-1 Murea at room temperature for 16 h. The column was washed with washbuffer (0.05 Na H₂ PO₄ 1 0.3 M Nacl, 10% glycerol) and tTF was elutedwith 0.2 M Imidozole in wash buffer. The eluted tTF was concentrated andloaded onto a G-75 column. tTF monomers were collected.

B. tTF

Gly[tTF]. The GlytTF complimentary DNA (cDNA) was prepared the same wayas described in the previous section except the 5' primer was replacedby the following primer in the PCR.

    5' primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG CTT CTG                                                        (SEQ ID NO:3)                                        GCA CTA CAA ATA CT                                              

The underlined sequence codes for the N-terminus of tTF. The remainingsequence encodes a restriction site for NcoI and a cleavage site forthrombin.

The H₆ pQE60 expression vector and the procedure for proteinpurification is identical to that described above except that the finalprotein product was treated with thrombin to remove the H₆ peptide. Thiswas done by adding 1 part of thrombin (Sigma) to 500 parts of tTF (w/w),and the cleavage was carried out at room temperature for 18 h. Thrombinwas removed from tTF by passage of the mixture through a BenzamidineSepharose 6B thrombin affinity column (Pharmacia).

C. Cysteine-modified tTFS

tTF constructs were modified with an N or C-terminal cysteine to allowfor easier conjugation to derivatized antibody through a disulfide bond.

H₆ C[tTF]. (His)₆ Ala Met Ala Cys-[tTF]. The DNA was made as describedin the previous section except that the 5' primer was replaced by thefollowing primer in the PCR.

    5' primer:  5' GTC ATG CCA TGG CCT GCT CAG GCA CTA CAA ATA                                                        (SEQ ID NO:4)                                       CTG TG                                                          

All of the procedures were the same as described above, except theN-terminal cys was protected with an exchangeable oxidizing/reducingreagent.

C[tTF]. Gly Ser Cys [tTF2-219]. The DNA was made as described in theprevious section except that the 5' primer was replaced by the followingprimer in the PCR.

    5' primer:  5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT                                                        (SEQ ID NO:5)                                       GCG GCA CTA CAA ATA CT                                          

The vector construct and protein purification is the same as describedfor the (His)₆ Ala Met Ala Cys [tTF] construct, except that thrombintreatment was used to remove the (His)₆ as described above.

H₆ [tTF]C. (His)₆ Ala Met Ala [tTF] Cys. The DNA was made the same wayas described in the (His)₆ AMA [tTF] sections, except that the 3' primerwas replaced by the following primer.

3' primer:5' TGA CAA GCT TAG CAT TCT CTG AAT TCC CCC TTT CT (SEQ IDNO:6)

The underlined sequence encodes the C-terminus of tTF. The rest of thesequence contains the HindIII restriction site for cloning tTF in to theexpression vector.

All of the procedures are the same as described in the tTF sectionexcept that 10 mM β-ME was used in the 6 M Gu-HCl denaturing solutionand the C-terminal cysteine was protected with an exchangeableoxidizing/reducing reagent.

Other [tTF] Cys monomers, such as [tTF 1-220] Cys, [tTF 1-221] Cys and[tTF 1-222] Cys are also made (and conjugated) using the samemethodology.

D. C Linker [tTF]

The C Linker [tTF], Gly-Ser-Cys-(Gly)₄ -Ser-(Gly)₄ -Ser-(Gly)₄-Ser-[tTF], was also constructed. The cDNA was made using a two step PCRprocedure as follows:

PCR 1: amplification of linker DNA

cDNA encoding the NcoI site, the thrombin cleavage site, cysteine,linker and the N-terminus of tTF was amplified using the followingprimers:

    5' primer:                                                                           5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT GCG                                                             (SEQ ID NO:7)                                       GA GGC GGT GGA TCA GGC                                              3' primer:                                                                           5' AGT ATT TGT AGT GCC TGA GGA TCC GCC ACC TCC                                                             (SEQ ID NO:8)                                       ACT                                                             

The underlined sequences encode the linker peptide. The DNA templateused in the PCR was double strand DNA encoding the following linker.

    Sequence:                                                                           GGA GGC GGT GGA TCA GGC GGT GGA GGT AGT GGA GGT                                                            (SEQ ID NO:9)                                     GGC GGA TCC                                                        

The same PCR conditions were used as described in the tTF section. The95 b.p. amplified product was linked to tTF DNA in the PCR2.

PCR 2: Linking the Cys-linker DNA to tTF DNA. DNA templates used in thePCR were two overlapping DNA: The 95 b.p. DNA from PCR 1 as describedabove and tTF DNA. The primers used were the following:

5' primer: 5' GTC ATG CCA TGG CCC TG (SEQ ID NO:10)

3' primer: 5' TGA CAA GCT TAT TCT CTG AAT TCC CCC TTT CT (SEQ ID NO:11)

The final PCR product of 740 b.p. was digested with NcoI and HindIII andinserted into the H₆ pQE 60 as described in the tTF section.

The vector constructs and protein purification procedures are all thesame as described in the C[tTF] section.

EXAMPLE V Synthesis of Dimeric Tissue Factor

The inventors' reasoned that tissue factor dimers may be more potentthan monomers at initiating coagulation. It is possible that nativetissue factor on the surface of J82 bladder carcinoma cells may exist asa dimer (Fair et al., 1987). The binding of one factor VII or VIIamolecule to one tissue factor molecule may also facilitate the bindingof another factor VII or VIIa to another tissue factor (Fair et al.,1987; Bach et al., 1986). Furthermore, tissue factor shows structuralhomology to members of the cytokine receptor family (Edgington et al.,1991) some of which dimerize to form active receptors (Davies andWlodawer, 1995). The inventors therefore synthesized TF dimers, asfollows.

A. [tTF] Linker [tTF].

The Gly [tTF] Linker [tTF] with the structure Gly[tTF] (Gly)₄ Ser (Gly)₄Ser (Gly)₄ Ser [tTF] was made. Two pieces of DNA were PCR amplifiedseparately and were ligated and inserted into the vector as follows:

PCR 1: Preparation of tTF and the 5' half of the linker DNA. The primersequences in the PCR are as follows:

    5' primer:                                                                             5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT                                                            (SEQ ID NO:12)                                  GCG GCA CTA CAA ATA CT                                                 3' primer:                                                                             5' CGC GGA TCC ACC GCC ACC AGA TCC ACC GCC TCC                                                            (SEQ ID NO:13)                                  TTC TCT GAA TTC CCC TTT CT                                         

Gly[tTF] DNA was used as the DNA template. Further PCR conditions wereas described in the tTF section.

PCR 2: Preparation of the 3' half of the linker DNA and tTF DNA. Theprimer sequences in the PCR were as follows:

    5' primer:  5' CGC GGA TCC GGC GGT GGA GGC TCT TCA GGC ACT                                                        (SEQ ID NO:14)                                 ACA AAT ACT GT                                                           3' primer:  5' TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T                                                          (SEQ ID NO:15)                        

tTF DNA was used as the template in the PCR. The product from PCR 1 wasdigested with NcoI and BamH. The product from PCR 2 was digested withHindIII and BamH1. The digested PCR1 and PCR2 DNA were ligated with NcoIand HindIII-digested H₆ pQE 60 DNA.

For the vector constructs and protein purification, the procedures werethe same as described in the Gly [tTF] section.

B. Cys [tTF] Linker [tTF]

The Cys [tTF] Linker [tTF] with the structure Ser Gly Cys [tTF 2-219](Gly)₄ Ser (Gly)₄ Ser(Gly)₄ Ser [tTF] was also constructed. DNA was madeby PCR using the following primers were used:

    5' primer:                                                                             5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT                                                            (SEQ ID NO:16)                                  GCG GCA CTA CAA ATA CT                                                 3' primer:                                                                             5' TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T                                                              (SEQ ID NO:17)                       

[tTF] linker [tTF] DNA was used as the template. The remaining PCO.conditions were the same as described in the tTF section. The vectorconstructs and protein purification were all as described in thepurification of H₆ C[tTF].

C. [tTF] Linker [tTF]cys

The [tTF] Linker [tTF]cys dimer with the protein structure [tTF] (Gly)₄Ser (Gly)₄ Ser (Gly)₄ Ser [tTF] Cys was also made. The DNA was made byPCR using the following primers:

    5' primer:                                                                             5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT GCA                                                              (SEQ ID NO:18)                                CTA CAA ATA CT                                                         3' primer:                                                                             5' TGA CAA GCT TAG CAT TCT CTG AAT TCC CCT TTC T                                                            (SEQ ID NO:19).                    

[tTF] linker [tTF] DNA was used as the template. The remaining PCRconditions were the same as described in the tTF section. The vectorconstructs and protein purification were again performed as described inthe purification of [tTF]cys section.

D. Chemically Conjugated Dimers

[tTF] Cys monomer are conjugated chemically to form [tTF] Cys-Cys [tTF]dimers. This is done by adding an equal molar amount of DTT to theprotected [tTF] Cys at room temperature for 1 hr to deprotect and exposethe cysteine at the C-terminus of [tTF] Cys. An equal molar amount ofprotected [tTF] Cys is added to the DTT/[tTF] Cys mixture and theincubation is continued for 18 h at room temperature. The dimers arepurified on a G-75 gel filtration column.

The Cys [tTF] monomer is conjugated chemically to form dimers using thesame method.

EXAMPLE VI Synthesis of Tissue Factor Mutants

Two tTF mutants are described that lack the capacity to converttTF-bound factor VII to factor VIIa. There is 300-fold less factor VIIain the plasma compared with factor VII (Morrissey et al., 1993).Therefore, circulating mutant tTF should be less able to initiatecoagulation and hence exhibit very low toxicity. In coaguligands, oncethe mutant tTF has localized through the attached antibody to the tumorsite, Factor VIIa will be injected to exchange with the tTF-bound FactorVII. The mutants are active in the presence of factor VIIa.

A. [tTF]G164A

The "[tTF]G164A" has the mutant protein structure with the amino acid164 (Gly) of tTF being replaced by Ala. The Chameleon double-strandedsite directed mutagenesis kit (Stratagene) is used for generating themutant. The DNA template is Gly[tTF] DNA and the sequence of themutagenizing primer is:

5' CAA GTT CAG CCA AGA AAAC (SEQ ID NO:20)

The vector constructs and protein purification procedures describedabove are used in the purification of Gly[tTF].

B. [tTF] W158R S162A

The [tTF]W158R S162A is a double mutant in which amino acid 158 (Trp) oftTF is replaced by Arg and amino acid 162 (Ser) is replaced by Ala. Thesame mutagenizing method is used as described for [tTF] G164A. Themutagenizing primer is:

5' ACA CTT TAT TAT CGG AAA TCT TCA GCT TCA GGA AAG (SEQ ID NO:21)

The foregoing vector constructs and protein purification procedures areused for purifying Gly[tTF].

EXAMPLE VII Synthesis of Tissue Factor Conjugates

A. Chemical Derivatization and Antibody Conjugation

Antibody tTF conjugates were synthesized by the linkage of chemicallyderivatized antibody to chemically derivatized tTF via a disulfide bond(as exemplified in FIG. 5).

Antibody was reacted with a 5-fold molar excess of succinimidyloxycarbonyl-α-methyl α-(2-pyridyldithio)toluene (SMPT) for 1 hour atroom temperature to yield a derivatized antibody with an average of 2pyridyldisulphide groups per antibody molecule. Derivatized antibody waspurified by gel permeation chromatography.

A 2.5-fold molar excess of tTF over antibody was reacted with a 45-foldmolar excess of 2-iminothiolane (2IT) for 1 hour at room temperature toyield tTF with an average of 1.5 sulfhydryl groups per tTF molecule.Derivatized tTF was also purified by gel permeation chromatography andimmediately mixed with the derivatized antibody.

The mixture was left to react for 72 hours at room temperature and thenapplied to a Sephacryl S-300 column to separate the antibody-tTFconjugate from free tTF and released pyridine-2-thione. The conjugatewas separated from free antibody by affinity chromatography on aanti-tTF column. The predominant molecular species of the finalconjugate product was the singly substituted antibody-tTF conjugate (Mrapprox. 176,000) with lesser amounts of multiply substituted conjugates(Mr≧approx. 202,000) as assessed by SDS-PAGE.

B. Conjugation of Cysteine-Modified tTF to Derivatized Antibody

Antibody-C[TF] and [tTF]C conjugates were synthesized by direct couplingof cysteine-modified tTF to chemically derivatized antibody via adisulfide bond (as exemplified in FIG. 5).

Antibody was reacted with a 12-fold molar excess of 2IT for 1 hour atroom temperature to yield derivatized antibody with an average of 1.5sulfhydryl groups per antibody molecule. Derivatized antibody waspurified by gel permeation chromatography and immediately mixed with a2-fold molar excess of cysteine-modified tTF. The mixture was left toreact for 24 hours at room temperature and then the conjugate waspurified by gel permeation and affinity chromatography as describedabove.

The predominant molecular species of the final conjugate was the singlysubstituted conjugate (Mr approx. 176,000) with lesser amounts ofmultiple substituted conjugates (Mr≧approx. 202,000) as assessed bySDS-PAGE.

C. Conjugation of Cysteine-Modified tTF to Fab° Fragments

Antibody Fab'-C[tTF] and [tTF]C conjugates are prepared. Such conjugatesmay be more potent in vivo because they should remain on the cellsurface for longer than bivalent conjugates due to their limitedinternalization capacity. Fab' fragments are mixed with a 2-fold molarexcess of cysteine-modified tTF for 24 hours and then the conjugatepurified by gel permeation and affinity chromatography as describedabove.

D. Clotting Activity of tTF Conjugates

tTF conjugates were prepared with the B21-2 monoclonal antibody whichbinds to Class II antigens expressed on the surface to A20 cells. Theconjugates were prepared with chemically derivatized tTF andcysteine-modified tTF and the ability of the conjugates to clot mouseplasma in CaCl₂ was determined after their binding to the surface of A20cells.

Both B21-2 conjugates shortened the clotting time of mouse plasma inCaCl₂ (control) in a dose-dependent manner. The tTF conjugates displayeda similar enhancement in coagulation as occurred when tTF was tetheredto the surface of A20 cells with the bispecific antibody B21-2/10H10(FIG. 6).

E. Anti-tumor Cell tTF Conjugates

It has already been established that when tTF is targeted to tumorvascular endothelial cells it induces coagulation within the tumorvessels (Examples I through III). The inventors' contemplated thatcoagulation would be induced in tumor vessels if tTF was targeted to thesurface of tumor cells.

Three anti-tumor cell antibodies, KS1/4, D612, and XMMCO-791, wereconjugated to tTF as described in the "Preparation of tTF conjugates"section above. KS1/4 was obtained from Dr. R. Reisfeld at the ScrippsResearch Institute, Department of Immunology, La Jolla, Calif., and isalso described in U.S. Pat. No. 4,975,369; D612 was obtained from Dr. J.Schlom at the NCI, Laboratory of Tumor Immunology and Biology, Bethesda,Md., is described in U.S. Pat. No. 5,183,756 and can be obtained fromculture supernatants from the ATCC hybridoma cell line Accession No. HB9796; XMMCO-791 was purified from tissue culture supernatant from thehybridoma cell line purchased from the ATCC.

The human colon carcinoma cell line Widr was used as a target cell forKS1/4. Widr cells were purchased from the ATCC and were maintained inDMEM supplemented with 10% (v/v) fetal calf serum, L-glutamine andantibiotics in an atmosphere of 10% (v/v) CO₂ in air. The human coloncarcinoma cell line LS147T was used as a target cell for D612. LS147Tcells were purchased from the ATCC and were maintained in RPMIsupplemented with 10% (v/v) fetal calf serum, L-glutamine andantibiotics in an atmosphere of 5% (v/v) CO₂ in air. The human non smallcell lung cancer cell line H460 was used as a target cell for XMMCO-791.H460 cells were obtained from Dr. Adi Gazdar, Simmons Cancer Center,University of Texas Southwestern Medical Center, Dallas, Tex. and weremaintained in DMEM supplemented with 10% (v/v) fetal calf serum,L-glutamine and antibiotics in an atmosphere of 10% (v/v) CO₂ in air.All three cell lines grew as adherent monolayers.

The conjugates were tested for their ability to enhance the clottingtime of mouse plasma in CaCl₂ when bound to tumor cells expressing therelevant target antigens. Tumor cells were removed from tissue cultureflasks with 0.05% (w/v) EDTA in PBS. The cells were preincubated withTF9-6B4 and TF8-5G9 antibodies to neutralize any native tissue factoractivity (Morrisey et. al., 1988) and then the coagulation assay wasperformed as described for A20 cells.

When bound to their target cell line, all three conjugates shortened theclotting time of mouse plasma in CaCl₂ (control) in a dose-dependentmanner (FIG. 7), indicating that coagulation was accelerated at thesurface of tumor cells when tTF was targeted to the cell surface.

EXAMPLE VIII Synthesis of Tissue Factor Prodrugs

Exemplary tTF prodrugs have the following structures: tTF₁₋₂₁₉ (X)_(n1)(Y)_(n2) Z Ligand, where tTF₁₋₂₁₉ represents TF minus the cytosolic andtransmembrane domains; X represents a hydrophobic transmembrane domainn1 amino acids (AA) in length (1-20 AA); Y represents a hydrophilicprotease recognition sequence of n2 AA in length (sufficient AA toensure appropriate protease recognition); Z represents a disulfidethioester or other linking group such as (Cys)₁₋₂ ; Ligand represents anantibody or other targeting moiety recognizing tumor-cells, tumor EC,connective tissue (stroma) or basal lamina markers

The tTF prodrug is contemplated for injection intravenously allowing itto localize to diseased tissue (i.e. tumor). Once localized in thediseased tissue, endogenous proteases (i.e., metalloproteinases,thrombin, factor Xa, factor VIIa, factor IXa, plasmin) will cleave thehydrophilic protease recognition sequence from the prodrug which willallow the hydrophobic transmembrane sequence to insert into a local cellmembrane. Once the tail has inserted into the membrane, the tTF willregain its coagulation-inducing properties resulting in clot formationin the vasculature of the diseased tissue.

EXAMPLE IX Synthesis of Coagulation Factors Lacking the Gla Modification

The vitamin-K-dependent coagulation factors (Factor II/IIa, FactorVII/VIIa, Factor IX/IXa and Factor X/Xa) lacking the Gla(γ-carboxyglutamic acid) modification are contemplated to be useful forthe formation of coaguligands. Coagulation factors lacking the Glamodification are poor coagulants because the unmodified factorsassociate inefficiently with lipid membranes: targeting the factor via aligand to the vasculature of tumors or other sites should bring thefactor back into proximity to the cell surface and enable coagulation toproceed in that site.

"Gla" is made post-translationally by modifying the existing Glu(Glutamic acid) residues. Vitamin-K-dependent coagulation factors(Factor II/IIa, Factor VII/VIIa, Factor IX/IXa and Factor X/Xa) lackingthe Gla modification may be made by expressing the genes that encodethem in a host, such as bacteria, that does not modify Glu to Gla. TheDNA sequences encoding each of Factor II/IIa, Factor VII/VIIa, FactorIX/IXa and Factor X/Xa are included herein as SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively. Procaryoticexpression is therefore straightforward.

Such Gla-lacking factors may also be made by mutating any of thesequences described above (SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 andSEQ ID NO:27) to change the corresponding Glu residues to another aminoacid before expressing the genes, this time in virtually any host cell.The codon to be changed is the GAG codon (GAA also encodes Glu and is tobe avoided). Using Factor VII as an example, the Gla "domain" is locatedgenerally in the 216-325 region. The first Gla-encoding triplet occursat 231 of SEQ ID NO:25, and the last extends through 318 of SEQ IDNO:25. The GAG codons may readily be changed using molecular biologicaltechniques.

FIG. 8 shows that the Gla domains of each of the abovevitamin-K-dependent coagulation factors lie in an analogous region.Therefore, mutation of the so-called "corresponding" Glu residues in anyone of SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:27 will also bestraightforward.

The following Table of codons is provided to enable ready mutationchoices to be made in modifying a given Gla-encoding codon or sequence.

    ______________________________________                                        Amino Acids    Codons                                                         ______________________________________                                        Alanine   Ala    A     GCA  GCC  GCG  GCU                                     Cysteine   Cys   C        UGC                                                                             UGU                                               Aspartic acid                                                                             Asp  D        GAC                                                                             GAU                                               Glutamic acid                                                                             Glu  E        GAA                                                                             GAG                                               Phenylalanine                                                                             Phe  F        UUC                                                                             UUU                                               Glycine     Gly  G        GGA                                                                             GGC   GGG  GGU                                    Histidine    His H        CAC                                                                             CAU                                               Isoleucine                                                                                Ile  I        AUA                                                                             AUC   AUU                                         Lysine       Lys K        AAA                                                                             AAG                                               Leucine     Leu  L        UUA                                                                             UUG   CUA  CUC   CUG                                                                               CUU                          Methionine                                                                                Met  M        AUG                                                 Asparagine                                                                                Asn  N        AAC                                                                             AAU                                               Proline     Pro  P        CCA                                                                             CCC   CCG  CCU                                    Glutamine    Gln  Q       CAA                                                                             CAG                                               Arginine   Arg   R        AGA                                                                             AGG   CGA  CGC   CGG                                                                               CGU                          Serine       Ser S        AGC                                                                             AGU   UCA  UCC   UCG                                                                               UCU                          Threonine    Thr T        ACA                                                                             ACC   ACG  ACU                                    Valine       Val  V       GUA                                                                             GUC   GUG  GUU                                    Tryptophan                                                                                Trp    W      UGG                                                 Tyrosine   Tyr   Y        UAC                                                                             UAU                                               ______________________________________                                    

Site-specific mutagenesis is the technique contemplated for use in thepreparation of individual vitamin-K-dependent coagulation factorslacking the Gla modification, through specific mutagenesis of theunderlying DNA and the introduction of one or more nucleotide sequencechanges into the DNA.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by publications such as Adelman et al. (1983)and by the TF mutant studies described above. The technique typicallyemploys a phage vector which exists in both a single stranded and doublestranded form. Typical vectors useful in site-directed mutagenesisinclude vectors such as the M13 phage (Messing et al., 1981). Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartthe two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the vitamin-K-dependentcoagulation factor. An oligonucleotide primer bearing the desiredmutated sequence is prepared, generally synthetically, for example bythe method of Crea et al. (1978). This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

EXAMPLE X Further Anti-tumor Vasculature Antibodies

This example describes the generation of antibodies directed againsttumor-derived endothelial cell "binding factors" for use indistinguishing between tumor vasculature and the vasculature of normaltissues. Particularly described is the generation of antibodies directedagainst vascular permeability factor (VPF), also termed vascularendothelial cell growth factor (VEGF), and against bFGF (basicfibroblast growth factor).

For further details concerning FGF one may refer to Gomez-Pinilla andCotman (1992); Nishikawa et al. (1992), that describe the localizationof basic fibroblast growth factor; Xu et al. (1992), that relates to theexpression and immunochemical analysis of FGF; Reilly et al. (1989),that concerns monoclonal antibodies; Dixon et al. (1989), that relatesto FGF detection and characterization; Matsuzaki et al. (1989), thatconcerns monoclonal antibodies against heparin-binding growth factor;and Herblin and Gross (1992), that discuss the binding sites for bFGF onsolid tumors associated with the vasculature.

In the present studies, rabbits were hyperimmunized with N-terminalpeptides of human VEGF, mouse VEGF, guinea pig VEGF, human bFGF, mousebFGF or guinea pig bFGF coupled to tuberculin (purified proteinderivative, PPD) or thyroglobulin carriers. The peptides were 25 to 26amino acids in length and were synthesized on a peptide synthesizer withcysteine as the C-terminal residue. Antisera were affinity purified oncolumns of the peptides coupled to Sephraose matrices.

Antibodies to VEGF were identified by ELISA and by their stainingpatterns on frozen sections of guinea pig tumors and normal tissues.Polyclonal antibodies to guinea pig VEGF and human VEGF reacted with themajority of vascular endothelial cells on frozen sections of guinea pigL10 tumors and a variety of human tumors (parotid, ovarian, mammarycarcinomas) respectively. The anti-human VEGF antibody stained mesangialcells surrounding the endothelial cells in normal human kidneyglomerulae and endothelial cells in the liver, but did not stain bloodvessels in normal human stomach, leg muscle and spleen. The anti-guineapig VEGF antibody did not stain endothelial cells in any normal tissues,including kidney, brain, spleen, heart, seminal vesicle, lung, largeintestine, thymus, prostrate, liver, testicle and skeletal muscle.

Polyclonal antibodies to human FGF stained endothelial cells in parotidand ovarian carcinomas, but not those in mammary carcinomas. Anti-humanFGF antibodies stained glomerular endothelial cells in human kidney, butnot endothelial cells in normal stomach, leg muscle and spleen.

Monoclonal antibodies to guinea pig VEGF, human VEGF and guinea pig bFGFwere prepared by immunizing BALB/c mice with the N-terminal sequencepeptides (with cysteine at the C-terminus of the peptide) coupled to PPDor to thyroglobulin. The synthetic peptides immunogens of definedsequence are shown below and are represented by SEQ ID NO:30, SEQ IDNO:31 AND SEQ ID NO:32, respectively:

    guinea pig VEGF                                                                         A P M A E G E Q K P R E V V K F M D V Y K R S Y C                   human VEGF                                                                              A P M A E G G G Q N H H E V V K F M D V Y Q R S Y C                 guinea pig bFGF                                                                         M A A G S I T T L P A L P E G G D G G A F A P G C               

The peptides were conjugated to thyroglobulin or to PPD by derivatizingthe thyroglobulin with succimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and reacting thederivative with the peptide. This yields a conjugate having one or morepeptide sequences linked via a thioether bond to thyroglobulin.

Specifically, the generation of monoclonal antibodies against the abovesequences was achieved using the following procedure: BALB/c mice wereimmunized by serial injections with peptide-PPD or peptide-thyroglobulininto several sites. Four or five days after the last injection, thespleens were removed and splenocytes were fused with P3xG3μg8.653myeloma cells using polyethyleneglycol according to the procedurespublished in Morrow, et al. (1991).

Individual hybridoma supernatants were screened as follows:

First screen: ELISA on peptide-thyroglobulin-coated plates.

Second screen: ELISA on cysteine linked via SMCC to thyroglobulin.

Third screen: Indirect immunoperoxidase staining of frozen sections ofguinea pig line 10 tumor or human parotid carcinoma.

Fourth screen: Indirect immunoperoxidase staining of frozen sections ofmiscellaneous malignant and normal guinea pig and human tissues.

Antibodies were selected that bound to peptide-thyroglobulin but not tocysteine-thyroglobulin, and which bound to endothelial cells inmalignant tumors more strongly than they did to endothelial cells innormal tissues (Table VIII).

                  TABLE VIII                                                      ______________________________________                                        Reactivity of Monoclonal Antibodies                                                        Reactivity with Tumor                                                                     Tumor                                                             Endothelium        Reactivity                                    MoAB  Immunogen.sup.+                                                                         Class  g. pig human  Pattern*                                 ______________________________________                                        GV14  gp VEGF   IgM    +      +      BV + some                                                                          tumor cells                         GV35       gp VEGF                                                                                                       Tumor cells                                                                  weak on BV                          GV39       gp VEGF                                                                                                       BV and some                                                                   tumor cells                        GV59       gp VEGF                                                                                                       BV and some                                                                   tumor cells                        GV97       gp VEGF                                                                                                       BV, weak on                                                                   tumor cells                        HV55       hu VEGF                                                                                                       Basement                                                                      membrane,                                                                     some BV                            GF67       gp FGF                                                                                                        BV and tumor                                                                  cells                              GFB2       gp FGF                                                                                                        BV and tumor                                                                 cells                               ______________________________________                                         *BV = blood vessels                                                           .sup.+ gp = guinea pig                                                        hu = human                                                               

A. GV97 Staining of Human and Guinea Pig Tissue Sections

The GV97 antibody against guinea pig VEGF N-terminus bound toendothelial cells in miscellaneous human malignant (Table IX) and normal(Table X) tissues. The GV39 and GV97 antibodies were deposited Dec. 12,1997, with the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md. 20852, U.S.A. and given the ATCC Accession numbersATCC HB 12450 and ATCC HB 12451, respectively. Effective Mar. 23, 1998,the address of the ATCC is: 10801 University Boulevard, Manassas, Va.,20110-2209, U.S.A.

Binding to endothelial cells in malignant tumors tended to exceed thatto endothelial cells in normal tissues.

The staining of endothelial cells in guinea pig tumor (line 10hepatocellular carcinoma) and normal tissues was similar in distributionand intensity to that observed with human tissues (Table XI).

In the Tables, +indicates a positive, as opposed to a negative, result.The numbers 2+, 3+ and 4+ refer to a positive signal of increasingstrength, as is routinely understood in this field of study.

                                      TABLE IX                                    __________________________________________________________________________    Anti-GPVEGF on Human Tumors                                                                  20 Purified GV97                                                                          1 ug/ml or                                                                         0.5 GV97         GV59                         Tumor  TISSUE  ug/ml                                                                            10 ug/ml                                                                           5 ug/ml                                                                           2 ug/ml                                                                            ug/ml                                                                             supt.                                                                              GV14                                                                              GV39                                                                              supt.                        __________________________________________________________________________    DIGESTIVE TRACT                                                               92-O1-A073                1+  2+                                                                           +/-                                              ve                          4++                                               esophagus carcinoma                                                           M4 Parotid                                    4+                              87-07-A134 Parotid;                                                                                         +/-                                             ve                          4+3+                                              carcinona                                                                     M5 Parotid                                                                    88-04-A010 parotid                                                                                     1+   1-2+                                            ve                                                                            ve                                  1-3+                                      adenoca.                                                                      90-11-B319 Adeno. Ca.                                                                                                              3-4+                     of colon to liver                                                             94-02-B021C                                      3-4+                         Adenocarcinoma of                                                             colon                                                                         93-10-A333 Adeno. Ca.                                                                                1-4++                                                  ve-1+                    4+                                                                                         3+                                      of colon with normal                                                          93-02-B004 Villous and                                                                                2-4+                                                                               1-2+                                                                                     3-4+                                                                                      2-3+                      Adenomatous polyp of                                                          colon                                                                         93-02-A130                      +/-1+                                         ve              4+                                                                                      4+                                                                                   3-4+                                         Leiomyosarcoma in                                                             colon                                                                         93-02-B020 Gastric Ca.                                                                                               3+                                     ve-1+                      1-2+                                                                           4+                                                93-04-A221 Pancreas                                                                                     2-3+4+                                                                            1-2+                                            ve-                             4+                                            Adenoca.                                 0.5+                                 94-04-A390 rectum                                                                                             1-2+                                                                                   1+                                                                                         3+                      adenoca.                                                                      93-12-A160 tongue                                                                                           +/-                                             ve                                                                            ve                          3+             3+                                 carcinomaadenoca.                                                             101-84a Stomach signet                                                        ve-1+                                                                         ve                most 1-                                                                                     3+                                            ring Ca. (101-84b                                                                                                       2+ but a                            pair)                                    few 3-4+                             90-05-A172 Stomach                                                                                           1-2+                                           ve-1+                                                                         ve               3+                                                           Adenoca.                                                                      REPRODUCTIVE TRACT                                                            91-10-A115 Sguam. cell                                                                       1-4+                                                                                1-3+                                                                             1-2+                                                                              1-2+                                                                                         1-4+                                                                                      1-3+                   Ca. of vulva                                                                  93-03-A343 Prostate                                                                                    +/- to +/- -                                                                       +/- to                                                                                /-        3-4+                                                                                3-4+                    Adenoca.                   2-3+  3-4+                                                                         1-2+                                          MUSCLE                                                                        MUNUNE SYSTEM                                                                 URINARY SYSTEM                                                                93-10-B002 Renal cell                                                                                             2+           3+                           Ca.                                                                           90-01-A225 Renal cll                                                                                            1-3+ of                                                                            3-4+                                                                                    3+                                                                                  3-4+                   Ca.                    of most                              some              93-01-A257 Transit.                                                                                  2-3+                                                                               +/--2+                                                                                   2-3+            2-3+                   cell Ca. of bladder                                                           ENDOCRINE SYSTEM                                                              94-01-A246                3-4+          4+                                                                  3+                                                                                      4+                                                                                     3-4+                         Pheochromocytoma of                                                           adrenal                                                                       93-11-A074 Adrenal                                                                              3-4+                                                                               3-4+                                                                                                     4+ 3-4+                     Cort. Ca.                                                                     RESPIRATORY SYSTEM                                                            93-08-N009 Lung                                                                                                            3-4+3-4+                                                                          3-4+                         Adenoca.                                                                      92-10-1016 Sq. cell                                                                                                 1-2+                                    ve-                         4++                                               lung Ca.                         0.5+                                         03-05-A065 Lung                                                               ve-1+               1+          3+                                                                                   3+                                     adenoca.                                                                      CENTRAL NERVOUS SYSTEM                                                        94-01-A299 malig.                                                                                                              3-4+                         metast. schwanoma to                                                          Lymph node                                                                    92-10-A139 Meningioma                                                                                                          3-4+                         91-12-A013 Meningioma                                                         ve-3+                             3+      4+                                  93-03-A361 Atypical                                                                                                      4+    3+                           meningioma                                                                    INTEGUMENTARY SYSTEM                                                          94-04-V037 Skin Sq.                                                           ve to                                                                         ve to                                                                         ve to 1+                                                                      ve                              2-3+        2-3+                              cell Ca. w/normal                                                                                         3+4+                                              89-02-225 leg sarcoma                                                                                       1+-4+                                                                                                   2+                    MISC. TUMORS                                                                  __________________________________________________________________________

                                      TABLE X                                     __________________________________________________________________________    Anti-GPVEGF on Human Normal Tissues                                                          20   Purified GV97                                                                             1 ug/ml or                                                                         0.5   GVP7           GV59                Tumor  TISSUE  ug/ml                                                                              10 ug/ml                                                                            5 ug/ml                                                                             2 ug/ml                                                                            ug/ml supt.                                                                              GV14 GV39 supt.               __________________________________________________________________________    DIGESTIVE SYSTEM                                                              91-01-A128                                                                    ve                              2-3+                                                                                2-3+                                    Bladder w/                                                                    cystitis                                                                      94-02-B020                                                                                                                               2-3+  2-3+         uninvolved colon                                                              92-01-A292 N.                              3-4+                                                                                            3-4+ 4+          Colon                                                                         93-10-A116 N.                                                                 ve-2+                                                                         ve                     3-4+                                                                                         3-4+                                    Colon                                                                         90-06-A116 N.                              3+         2+                      colon                                      of many                            93-02-A350 N.                                   +/-                                                                                     4+                  esophagus                                                                     93-05-A503 N.                                                                                                                           4+  4+              Ileum                                                                         94-03-A244 N.            4+                                                   ve-1+                                                                         ve                                         4+                                 Liver                                                                         90-02-B132 N.                                                                 ve                                                                            ve                                                                            ve                       2-3+                                                                               2-3+                                                                                  2-3+                                    Liver                           a few                                         94-01-A181 N.                               of                                ve                                                                                                            3-4+                                          Pancreas                                                                                                      a few                                         90-05-D008 N.        2-4+                                                     ve                                    2-3+           2-3+                     Pancreas                                                                      93-05-A174 N.                1-2+ of 2+ of                                                                      1+ of                                       ve                                                                            ve                       3+ of                                                                             2-3+                                             Parotid                                   a few      a few                    94-04-A391 N.                        1-3+                                     ve-2+                                                                         ve                                                                            ve                               3+                                           Small bowel                                                                   88-06-107 N.                                                                  ve                                  3-4+                                                                           3+                                       Stomach                                                                       101-84b N.                  2-3+ in       3-4+                                                                    +/- in                                    ve in main         3+                                      3-4+               Stomach (101=84a                                                                                                  main and                                                                            and 1+ in                           pair)                           3-4+ iny                                                                       2+  in                                                                                   periphery                                                    periphery                                                                          periphery                                     90-11-B337 N.                                                                 ve                                                                            ve                                          3+                                Stomach                                                                       REPRODUCTIVE TRACT                                                            93-04-A041 N.                                        3+      4+               Breast                                                                        94-02-A197 N.                                                                                                             4+       3+                       Breast                                                                        w/fibrocystic                                                                 change                                                                        93-02-A051 Breast                                                             ve-1+                                                                         ve                                                                            ve                                                                            2+                                 +/-                 +/-                    w/fibrocystic                                                                 change                                                                        93-02-A103 Breast                                                                                              1+   2+                                      w/fibrocyst.                                                                  change                                                                        92-11-A006 N.        1-2+ of                                                                                0.5+                                            ve                                                                            ve                   1-2+ of                            3+of                  ectocervix     most of most                                                                                                                  most  some     91-03-A207 N.                                      5+                                                                                   2-3+                ectocervix                                                                    92-02-A139 N.          1+ in most                                             ve in most                                                                    ve                                                                            ve                                                                            ve in                                                                         ve in                                                                         ovary w/corp.          but 1+ in                                                                                                             most           lusteum                  one arearea                                                                                                           bet 3-4                                                               ar                                                                             in one                                                                               area         93-06-A11B N.                         1+ of a few                             ve                                                                            ve                                                                            ve                              3+                                            Prostate                                                                      93-11-A317d                               4+                                  ve-3+                                                                         ve-1+                                 3-4+            3-4+                    Prostate chip                                                                 93-02-A315                                 -1+                                ve                                                                            ve                                    1.2+             1+                     Seminal Vesicle                                                               92-04-A069 N.             1+        +/-                                                                                     +/--                                                                                     1-2+                 testis                                                                        91-04-A117 Ureter                                                                                 1+        +/-                                             ve                                                                            1+                  3-4+                            +/-                       w/inflammation                                                                MUSCLE                                                                        94-01-A065 N.         3-4+                                                                                2+              +/-                               ve                               4+                 3-4+                      Heart                                                                         91-07-D007 N.         1-3+1-4+                                                                                    1-2+                                      ve                                                                            ve                 1-3+                                  1-3+                 skeletal muscle                                                               95-03-A395 N.          4+       3-4+                                                                                        0.5-1+                                                                                     4++                Skeletal muscle                                                               IMMUNE SYSTEM                                                                 90-01-A077 N.           2+2-3+                                                                                       some                                   ve                                                                            ve                     2-3+                             3-4+                  lymph node                                                                    90-08-A022 N.              most 0, 5++ but                                                                       most -ve                                                                           most -ve                                                                                        3+                  lymph node                    but a few+                                                                           but a few                                                                            but a few                                                                               0.5-1+                  91-03-A057 N.                                                                 ve                                    3-4+          3-4+                      lymph node                                                                    1+-09-B017lE                              +/-                                 ve                                    2-3+           2-3+                     uninvolved lymph                                                              node                                                                          93-07-A236 N.                               3-4+                              ve-3+                                                                         ve                                                                   2-4+     Spicen                                                                        93-07-252 N.                                                                  ve                                                  2-3+                      spicen                                                                        ENDOCRINE SYSTEM                                                              94-04-A252 N.                             1-2+                                                                                           3+   4+            adrenal w/                                                                    medulia and                                                                   cortex                                                                        93-05-A086 N.              most -ve a-ve a                                    ve                                                                            ve                                    3-4+          2-3+                      Adrernal medulla                                                                                             few 1-2++                                      92-03-A157                         +/-  +/-                                   ve                        4+     4+                                           Hyperplasic                                                                   thyroid                                                                       91-03-B019 N.                                                                 ve-3+                                                                         ve-2+                                                                         ve-1+                                                                         ve                                    2-3+         2-3+                       Thyroid                                                                       URINARY SYSTEM                                                                93-09-A048 N.                                                                                                                           2-3+                Kidney                                                                        91-11-A075 N.                                       4+ on                                                                                    4+ onn         Kidney                                     glomeruli glomeruli                                                                          glomeruli           93-10-B001 N.                                 -                               ve                     4+ on                                                                                  4+ on 4+ on                                   Kidney                                                                                                                   glomeruli      glomeruliuli        INTEGUMENTARY SYSTEM                                                          92-08-A029 N.                  +/- to 3+- to 4+                                                                            /-                                                                                                 2+          Breast skin                                                                   89-02-257                                      1-2+                                                                                            3-4+         Cartiledge                                                                    marches 2SS                                                                   RESPIRATORY SYSTEM                                                            93-05-A203 N.                                                                 ve-2+                                                                         ve-1+                                                                         ve                               3+                    2+                     Lung                                                                          92-12-A263 N.             2-3+ w/                                                                           1-2+  w/                                        ve                                                                            ve                                                                 2-3+       Bronchus                       ducts                                                                staining                                                                                               staining                                           3-4+                        2-3+                          __________________________________________________________________________

                                      TABLE XI                                    __________________________________________________________________________    Staining Pattern of 9F7 anti-VEGF by direct immunohistochemical               staining on 6-8 week old GP tissues                                                               Purified GV97 1 ug/ml or                                  TISSUE        20 ug/ml                                                                            10 ug/ml                                                                             5 ug/ml                                                                              2 ug/ml                                                                             0.5 ug/ml                                                                           9F7 supt.                                                                          3F9 supt.                                                                           5F9                  __________________________________________________________________________                                                             supt.                DIGESTIVE SYSTEM                                                              LIVER                             1-2+                                                                             +/-                                                                                 +/-                                                                                            1-2+              INTESTINE                 3+                                                                                                              4 + m                                                                lymphoi                                                                              lymphoid,                                                                               rest                                                         diff.  rest diff.                                                             than         than          PANCREAS              1+ of many                                                                            and 3+ in                                                                      islands of                                                                         cells                                     SMALL                2-3+ of manyny                                                                         1-2+ of                                                                               +/- of many                                                                                     3+ of                                                                          3+ of some           INTESTINE                and 4+ in                                                                          many and 4+                                                                            and 4+ in                                                                                      an                                                                               and 4+ in                                                                   l                                                                                   lymphoid                                      lymphoid,                 rest                                                     rest diff.                                                              rest diff.                 than                                                      than fVIII                                                               than fVIII                                    STOMACH                    1-2+ on most                                                                   +/- on most                                                                          +/- on most                    3-4+                                                           (some 3-4+ (some                                         occasionalccasional                                                                  occasional                                                                                          fVIII-ve)-ve)                                                1+                 3+               REPRODUCTIVE SYSTEM                                                           TESTIS                                                                        MUSCLE AND INTEGUMENTARY SYSTEM                                               HEART                                                                         ve                                                                            ve                                                                            ve                                                                            ve                                                                                                                               3-4+ (some                                                                          3-4+ (some                                                              fVIII-v                                                                               fVIII-ve)          MUSCLE                                                                        SKIN                    1+ in fatty                                                                           +/- in                                                                               +/- in fatty                                                                                         3++                                       layer and 3-tty                                                                  fatty layer                                                                          layer and 1-                                                        4+ in    layer and                                                                   and 3-4+ of                                                                      2+ of a few                                                         cellular  3-4+ in                                                                   a few in                                                                            in cellular                                                        layer   cellular                                                                     cellular                                                                             layer                                                                        layer                                 IMMUNE SYSTEM                                                                 SPLEEN                                                                        ve                         4+     4+                                          THYMUS                                                                        URINARY SYSTEM                                                                KIDNEY                 glomeruli 3-                                                                        glomeruli                                                                             glomeruli 1-     glom                                                                                glomeruli                                              2++                                                                                                  3-4+              ENDOCRINE SYSTEM                                                              ADRENAL                                                                       RESPIRATORY SYSTEM                                                            LUNG                                                                          NERVOUS SYSTEM                                                                CEREBELLUM                        +/- of most                                                                      +/- of most                                                                                           4+                                             and 1+ of a                                                                          and 1+ of a                                                                           few                              TUMORS                                                                        TUMOR                   4+4+                                                                                 3-4+                                                                                  2-3+ (2)                                                                               4+       3+                   __________________________________________________________________________

B. Lack of Reactivity of GV97 With Soluble Human VEGF

To identify antibodies that are specific for VEGF, the VEGF receptor(Flk-1) or VEGF bound (or complexed) to the receptor, an ELISA screeningprotocol was developed. The procedure is as follows:

Initially, a 96 well ELISA plate (round bottom) was coated (outsidewells left blank) with 100 μl/well of FLK/seap at 10 μg/ml insensitizing buffer. After overnight incubation, the plate was washedtwice with PBS overnight at 4° C. Next the FLK/Seap coated plate wasblocked with 250 μl/well of PBS+CAH (5%) solution for 1 h at 37° C. Theblocking solution was removed and the plate was vigorously tapped onpaper towels.

The blocked plates were then incubated with 100 μl/well of VEGF-165(VEGF 165 aa form produced in yeast obtained from Dr. Ramakrishnan,University of Minnesota) at 2 μg/ml in binding plus 0.1 μg/ml heparinfor 4 h at room temperature or overnight at 4° C. The VEGF solution wascollected and the plate washed 2 times with PBS-tween (0.10%). Next, 100μl/well of hybridoma fusion supernatant was added to the wells andincubated for 1 h at 32° C. Following this supernatant incubation, theplate was washed 3 times with PBS tween and then incubated with 100 μlwell of secondary antibody (KPL, Gt anti-mouse IgG at 1:1000 in PBStween+CAH (5%) for 1 hour at 37°C.

Following secondary antibody incubation, the plates were washed 4 timeswith PBS tween, incubated with 100 μl/well of substrate (Substrate SigmaOPD dissolved in citrate buffer+H₂ O₂) for 20 minutes and read at 490 nmon a Cambridge Technology Microplate Reader (Model 7520). Wells with anabsorbance above appropriate control wells were selected as positivesand further characterized.

It was found that GV97 did not bind to recombinant VEGF-coated ELISAplates, nor did recombinant human VEGF bind to GV97 coated ELISA plates.Soluble recombinant human VEGF did not block the binding of 5 μg/ml GV97to tumor endothelium in histological sections even when added at 50μg/ml.

These data suggest that GV97 recognizes an epitope of VEGF that isconcealed in recombinant human VEGF but which becomes accessible whenVEGF binds to its receptor on endothelial cells.

C. GV97 Localization in Line 10-Bearing Guinea Pigs

In contrast with staining data obtained from histological sections, GV97antibody localized selectively to tumor endothelial cells afterinjection into line 10 tumor-bearing guinea pigs (Table XII). Stainingof endothelial cells in the tumor was moderately strong whereas stainingof normal endothelium in miscellaneous organs was undetectable.

D. Anti-bFGF Selectively Bind to Tumor Endothelial Cells

GV97 and GF82, which had been raised against guinea pig bFGF N-terminus,bound strongly to endothelial cells in frozen reactions of guinea pigline 10 tumor and to endothelial cells in two types of human malignanttumors (Table XIII). By contrast, relatively weak staining ofendothelial cells in miscellaneous guinea pig normal tissues wasobserved.

                  TABLE XII                                                       ______________________________________                                        GV97 injected into tumor bearing GP                                                                      GV 97 20 ug/ml serum                               TISSUE        GV97 10 ug/ml                                                                                       volume injected                           ______________________________________                                        DIGESTIVE SYSTEM                                                              LIVER       2+             -ve                                                INTESTINE        3+         possible 0.5-1+ of a                                                           few                                              PANCREAS      +/- of many and 2+ in                                                                       possible 0.5-1+ of a                                            islands of cells                                                                               few                                            SMALL INTESTINE                                                                             2-3+ of many and 4+ in                                                                     +/-                                                            lymphoid, rest diff.                                                           than fVIII                                                       STOMACH       1-2+ on most occasional                                                                    possibly 0.5+ of a few                                            3+                                                             REPRODUCTIVE SYSTEM                                                           TESTIS                     +/-                                                MUSCLE AND INTEGUMENTARY SYSTEM                                               HEART       -ve            -ve                                                MUSCLE                        -ve                                             SKIN            1+ in fatty layer and                                                       3-4+ in cellular layer                                          IMMUNE SYSTEM                                                                 SPLEEN      3+             possibly a few 1+                                  THYMUS                                                                        URINARY SYSTEM                                                                KIDNEY      glomeruli 3-4+                                                    ENDOCRINE SYSTEM                                                              ADRENAL     4+             -ve                                                RESPIRATORY SYSTEM                                                            LUNG        2+             -ve                                                NERVOUS SYSTEM                                                                CEREBELLUM  2+             -ve                                                TUMORS                                                                        TUMOR       4+             2-3+                                               ______________________________________                                    

                  TABLE XIII                                                      ______________________________________                                        Anti-GP FGF Antibody Staining on GP Tissues                                   GP TISSUE           GF 67   GF 82                                             ______________________________________                                        DIGESTIVE SYSTEM                                                              LIVER               ND      ND                                                INTESTINE               +/- +/-                                               PANCREAS                 2-3+                                                                                 2+                                            SMALL INTESTINE          +/-                                                                                     +/-                                        STOMACH                   ND                                                                                   ND                                           REPRODUCTIVE SYSTEM                                                           TESTIS              ND      ND                                                MUSCLE AND INTEGUMENTARY SYSTEM                                               HEART               2-3+    1+                                                MUSCLE                     +/-                                                                            1+                                                SKIN                     ND      ND                                           IMMUNE SYSTEM                                                                 SPLEEN              3+      -ve                                               THYMUS                                                                        URINARY SYSTEM                                                                KIDNEY              1-2+    -ve                                               ENDOCRINE SYSTEM                                                              ADRENAL             1-2+    +/-                                               RESPIRATORY SYSTEM                                                            LUNG                1-2+    2-3+                                              NERVOUS SYSTEM                                                                CEREBELLUM          1+      -1+                                               TUMORS                                                                        LINE 1 TUMOR        4+      4+                                                HUMAN TUMORS                                                                  PHEOCHROMO CYTOMA   4+      4+                                                SCHWANOMA               4+     4+                                             ______________________________________                                    

EXAMPLE XI Human Treatment Protocols

This example is concerned with human treatment protocols using thebispecific binding and coagulating ligands of the invention. Theseligands are contemplated for use in the clinical treatment of varioushuman cancers and even other disorders, such as benign prostatichyperplasia and rheumatoid arthritis, in which the intermediate orlonger term arrest of blood flow would be advantageous.

The bispecific ligands are considered to be particularly useful tools inanti-tumor therapy. From the data presented herein, including the animalstudies, and the knowledge in the art regarding treatment of Lymphoma(Glennie et al., 1988), T-Cell targeting (Nolan & Kennedy, 1990) anddrug targeting (Paulus, 1985) appropriate doses and treatment regimensmay be straightforwardly developed.

Naturally, before wide-spread use, further animal studies and clinicaltrials will be conducted. The various elements of conducting a clinicaltrial, including patient treatment and monitoring, will be known tothose of skill in the art in light of the present disclosure. Thefollowing information is being presented as a general guideline for usein establishing such trials.

It is contemplated that patients chosen for the study would have failedto respond to at least one course of conventional therapy and had tohave objectively measurable disease as determined by physicalexamination, laboratory techniques, or radiographic procedures. Wheremurine monoclonal antibody portions are employed, the patients shouldhave no history of allergy to mouse immunoglobulin. Any chemotherapyshould be stopped at least 2 weeks before entry into the study.

In regard to bispecific ligand administration, it is considered thatcertain advantages will be found in the use of an indwelling centralvenous catheter with a triple lumen port. The bispecific ligands shouldbe filtered, for example, using a 0.22 μm filter, and dilutedappropriately, such as with saline, to a final volume of 100 ml. Beforeuse, the test sample should also be filtered in a similar manner, andits concentration assessed before and after filtration by determiningthe A₂₈₀. The expected recovery should be within the range of 87 to 99%,and adjustments for protein loss can then be accounted for.

The bispecific ligands may be administered over a period ofapproximately 4-24 hours, with each patient receiving 2-4 number ofinfusions at 2-7 day intervals. Administration can also be performed bya steady rate of infusion over a 7 day period. The infusion given at anydose level should be dependent upon any toxicity observed. Hence, ifGrade II toxicity was reached after any single infusion, or at aparticular period of time for a steady rate infusion, further dosesshould be withheld or the steady rate infusion stopped unless toxicityimproved. Increasing doses of bispecific coagulating ligands should beadministered to groups of patients until approximately 60% of patientsshowed unacceptable Grade III or IV toxicity in any category. Doses thatare 2/3 of this value could be defined as the safe dose.

Physical examination, tumor measurements, and laboratory tests should,of course, be performed before treatment and at intervals up to 1 monthlater. Laboratory tests should include complete blood counts, serumcreatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT,bilirubin, albumin, and total serum protein. Serum samples taken up to60 days after treatment should be evaluated by radioimmunoassay for thepresence of the intact bispecific ligand or components thereof andantibodies against any or both portions of the ligand. Immunologicalanalyses of sera, using any standard assay such as, for example, anELISA or RIA, will allow the pharmacokinetics and clearance of thetherapeutic agent to be evaluated.

To evaluate the anti-tumor responses, it is contemplated that thepatients should be examined at 48 hours to 1 week and again at 30 daysafter the last infusion. When palpable disease was present, twoperpendicular diameters of all masses should be measured daily duringtreatment, within 1 week after completion of therapy, and at 30 days. Tomeasure nonpalpable disease, serial CT scans could be performed at 1-cmintervals throughout the chest, abdomen, and pelvis at 48 hours to 1week and again at 30 days. Tissue samples should also be evaluatedhistologically, and/or by flow cytometry, using biopsies from thedisease sites or even blood or fluid samples if appropriate.

Clinical responses may be defined by acceptable measure. For example, acomplete response may be defined by the disappearance of all measurabletumor 1 month after treatment. Whereas a partial response may be definedby a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules 1 month aftertreatment, with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater 1 month aftertreatment, with progression in one or more sites.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Abbassi et al., J Clin Invest, 92(6):2719-30, 1993.

Abraham et al., Science, 233:545-548, 1986.

Abrams & Oldham, Monoclonal antibody therapy of human cancer, Foon &Morgan (Eds.), Martinus Nijhoff Publishing, Boston, pp. 103-120, 1985.

Adams et al., Cancer Res., 43:6297, 1983.

Adelman et al., DNA 2:183, 1983.

Alvarez e t al., Modern Pathology, 5(3):303-307, 1992.

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Arklie et al., Int. J. Cancer, 28:23, 1981.

Ashall et al., Lancet, 2(8288):7-10, 1982.

Atkinson et al., Cancer Res., 62:6820, 1982.

Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y., 1989.

Bach et al., Biochemistry, 25, 4007-4020, 1986.

Bauer et al., Vox Sang, 61:156-157, 1991.

Baxter et al., Micro. Res., 41(1):5-23, 1991.

Bevilacqua et al., Proc. Natl. Acad. Sci. USA, 84:9238-9242, 1987.

Bhagwa et al., Nature, 316:511-513, 1985.

Bhattacharya et al., Hybridoma, 4:153, 1985.

Bhattacharya et al., Cancer Res., 44:4528, 1984.

Bicknell and Harris, Seminars in Cancer Biology, 3:399-407, 1992.

Bikfalvi et al., Exp. Cell Res., 181:75-84, 1989.

Birembaut et al., J. Pathology, 145:283-296, 1985.

Bittner et al., Methods in Enzymol., 153:516-544, 1987.

Bjorndahl et al., Eur. J. Immunol., 19:881-887, 1989.

Blakey et al., Biochem Biophys ACTA, 923Y(1):59-65, 1987b.

Blakey et al., Cancer Res., 47:947-952, 1987a.

Bolhuis et al., J. Immunol., 149:1840-1846, 1992.

Borden et al., Cancer, 65:800-814, 1990.

Brennan et al., Science, 229:81-83, 1985.

Brinkmann et al., Proc. Natl. Acad. Sci., 88(19):8616-20, 1991.

Brooks et al., Cell, 79:1157-1164, 1994.

Brooks et al., Science, 264:569-571, 1994.

Brown et al., J. Exp. Med., 176:1375-1379, 1992.

Brown et al., PNAS, 78:539, 1981a.

Brown et al., J. Immunol., 127:539, 1981b.

Brown et al., Cancer Res., 53:4727-4735, 1993.

Broze, Seminars in Hematol., 29:159-169, 1992.

Bruland et al., Cancer Research, 48:5302-5309, 1988.

Bruland et al., Int. J. Cancer, 38(1):27-31, 1986.

Buhring et al., Leukemia, 5:841-847, 1991.

Burchell et al., J. Immunol., 131(1):508-13, 1983.

Burrows, & Thorpe, PNAS, 90:8996-9000, 1993.

Burrows et al., Cancer Res, 52:5954-5962, 1992.

Burrows et al., Cancer Res., 51:4768-4775, 1991.

Burrows et al., Clin. Cancer Res., 1995 (in press).

Burtin et al., Cancer, 31:719-726, 1983.

Byers & Baldwin, Immunol, 65:329-335, 1988.

Byers et al., Cancer Res., 49:6153-6160, 1989.

Byers et al., 2nd Int. Conf. Mab Immunocon., Cancer, 41:1987.

Campbell, In: Monoclonal Antibody Technology, Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13, Burden & Von Knippenberg(Eds.), Elseview, Amsterdam, pp. 75-83, 1984

Carnemolla et al., J. Cell Biol., 108:1139-1148, 1989.

Carnemolla et al., J. Biol. Chem., 267(34):24689-24692, 1992.

Carrel et al., Hybridoma, 1:387, 1982.

Cavenagh et al., Br J Haematol, 85(2):285-91, 1993.

Chapman et al., Arthritis Rheum, 37(12):1752-6, 1994.

Chee et al., Cancer Res., 43:3142, 1982.

Chen et al., J. Immunol, 145:8-12, 1990.

Cherwinski et al., J. Exp. Med., 166:1229-1244, 1989.

Cheung et al., Proc. AACR, 27:318, 1986.

Clark et al., Biochim. Biophys. ACTA, 867:244-251, 1986.

Clark et al., Cancer Res., 51:944-948, 1991.

Clark et al., Int. J. Cancer, 2:15-17, 1988.

Clauss et al., J. Exp. Med., 172:1535-1545, 1990.

Cohn et al., Arch. Surg., 122:1425, 1987.

Colberre-Garapin et al., J. Mol. Biol., 150:1, 1981.

Colcher et al., Cancer Invest, 1:127, 1983.

Colcher et al., Cancer Res., 47:1185 and 4218, 1987.

Colcher et al., PNAS, 78:3199, 1981.

Collins et al., Proc. Natl. Acad. Sci. U.S.A., 81:4917-4921, 1984.

Conn et al., Proc. Natl. Acad. Sci. USA, 87:2628-2632, 1990.

Connolly et al., J. Biol. Chem., 264(33):20017-20024, 1989.

Corgon-Cardo et al., Laboratory Investigation, 63(6):832-840, 1990.

Corvalen, Cancer Immuno., 24:133, 1987.

Cotran et al., J. Exp. Med., 164:661-666, 1986.

Crea et al., Proc. Natl. Acad. Sci. U.S.A 75:5765, 1978.

Croghan et al., Cancer Res., 43:4980, 1983.

Croghan et al., Cancer Res., 44:1954, 1984.

Daar et al., Transplantation, 38(3):293-298, 1984.

Davies and Wlodawer, FASEB J., 9:50-56, 1995.

Davis & Preston, Analytical Biochemistry, 116(2):402-407, 1981.

de Krester et al., Int. J. Cancer, 37:705, 1986.

De Vries et al., Science, 255:989-991, 1992.

DeFranco, Nature, 352:754-755, 1991.

deLeij et al., Bispecific antibodies and targeted cellular cytotoxicity,Romet-Lemonne et al., p. 249, 1991.

Denekamp, et al., Brit. J. Cancer, 461:711-720, 1982.

Denekamp, Cancer Meta. Rev., 9:267-282, 1990.

Denekamp, Prog. Appl. Microcirc., 4:28-38, 1984.

Detmar et al., J. Exp. Med., 180:1141-1146, 1994.

Dewerchin et al., Blood, 78(4):1005-1018, 1991.

Di Scipio et al., Biochemistry, 16:5253-5260, 1977.

Dillman et al., Antibody, Immunocon. Radiopharm., 1:65-77, 1988.

Dippold et al., PNAS, 77:6115, 1980.

Dixon et al., Mol. & Cell Biol., 7:4896-4902, 1989.

Duijvestijn et al., J. Immunol., 138:713-719, 1987.

Dunham & Stewart, J. Natl. Cancer Inst., 13:1299-1377, 1953.

Dustin et al., J. Immunol., 137:245-254, 1986.

Dvorak et al., J. Exp. Med., 174:1275-1278, 1991.

Dvorak et al., Cancer Cells, 3(3):77-85, 1991.

Edelman et al., Proc. Natl. Acad. Sci. USA, 90:1513-1517, 1993.

Edgington et al., Thrombosis and Haemostasis, 66(1):67-79, 1991.

Ellis et al., Histopathol., 8:501, 1984.

Embleton et al., Br. J. Cancer, 63(5):670-674, 1991.

Epenetos et al., Cancer Res., 46:3183-3191, 1986.

Epenetos et al., Lancet, Nov. 6, 2:1000-1004, 1982.

Fair et al., J. Biol. Chem., 262, 11692-11698, 1987.

Farrans et al., Lancet, 2:397, 1982.

Febbraio and Silverstein, J. Biol. Chem., 265(30):18531-18537, 1990.

Ferrara et al., J. Cell. Biochem., 47:211-218, 1991.

Ferrara et al., Endocrine Reviews, 13(1):18-32, 1992.

Fisher et al., Thrombosis Research, 48:89-99, 1987.

Flavell et al., Br. J. Cancer, 65:545-551, 1992.

Flavell et al., Br. J. Cancer, 64(2):274-280, 1991.

Folkman, Adv. Cancer Res., 43:175-230, 1985a.

Folkman et al., Ann. Surg., 214(4):414-427, 1991.

Folkman, In: Important Advances in Oncolory, Part I, DeVita et al.(Eds.), JB Lippincott, Philadelphia, pp. 42-62, 1985b.

Foster et al., Virchows Arch. (Pathol. Anat. Histophatol.), 394:295,1982.

Foster et al., Human Pathol., 15:502 1984.

Fox, et al., J. Biol. Resp., 9:499-511, 1990.

Frelinger III et al., J. Biol. Chem., 266(26):17106-17111, 1991.

Frelinger III et al., J. Biol. Chem., 265(11):6346-6352, 1990.

French et al., Cancer Res., 51:2358-2361, 1991.

Gailani and Broze, Jr., Science, 253:909-912, 2991.

Galfre et al., Methods Enzymol., 73:1-46, 1981.

Gallagher et al., J. Surg. Res., 40:159, 1986.

Galland et al., ???????, 1233-1240, 1993.

Gangopadhyay et al., Cancer Res., 45:1744, 1985.

Gefter et al., Somatic Cell Genet., 3:231-236, 1977.

Geppert et al., Immunological Reviews, 117:5-66, 1990.

Ghetie et al., Cancer Res., 51:5876-5880, 1991.

Ghose, CRC Critical Review in Therapeutic Drug Carrier Systems,3:262-359, 1982.

Ghose & Blair, CRC Critical Reviews in Therapeutic Drug Carrier Systems,3:262-359, 1987.

Gibbons, In: J. D. Gibbons (ed.), "Nonparametric methods forquantitative analysis," pp. 160, New York: Holt, Rinehart and Winston.1976.

Gitoy-Goren et. al., Biochem. Biophys. Res. Comm., 190:702-, 1993

Glassy et al., PNAS, 80:63227, 1983.

Glennie et al., J. Immunol., 141(10):3662-3670, 1988.

Glennie et al., J. Immunol., 139:2367-2375, 1987.

Goding, In: Monoclonal Antibodies: Principles and Practice, 2d ed.,Academic Press, Orlando, Fla., pp. 60-61, 65-66, 71-74, 1986.

Gomez-Pinilla and Cotman, Neuroscience, 49:771-780, 1992.

Gosset et al., Int Arch Allergy Immunol, 106(1):69-77, 1995.

Gougos et al., Int. Immunol., 4:83-92, 1992.

Gougos & Letarte, J. Immunol., 141:1925-1933, 1988.

Griffin et al., Treat. Res., 37:433-455, 1988b.

Griffin et al., Proc. 2nd Conf. on Radioimmunodetection & Therapy ofCancer, 82, 1988a.

Groenewegen et al., Nature, 316:361-363, 1985.

Groves et al., Br J Dermatol, 124(2):117-23, 1991.

Gusterson et al., Br. J. Cancer, 58:453, 1988.

Hagemeier et al., Int. J. Cancer, 38:481-488, 1986.

Hakkert et al., Blood, 78(10):2721-6, 1991.

Hammerling, Transplant. Rev., 30:64-82, 1976.

Hattey et al., Thrombosis Research, 45(5):485-495, 1987.

Hayes et al., J. Clin. Invest., 75:1671, 1985.

Hayward et al., Biological Chemistry, 266(11):7114-7120, 1991.

Hendler et al., Trans. Assoc. Am. Physicians, 94:217, 1981.

Herblin and Gross, Angiogenesis: KeyPrinciples--Science--Technology--Medicine, 214-218, 1992.

Hess et al., Transplantation, 6:1232-1240, 1991.

Heynen et al., J. Clin. Invest., 94:1098-1112, 1994.

Horan Hand et al., Cancer Res., 45:2713, 1985.

Howard et al., Developmental Biology, 146:325-338, 1991.

Huang et al., Anticancer Research, 13:887-890, 1993.

Imai et al., JNCI, 68:761, 1982.

Imam et al., J. Immunobiol, 1984.

Inouye et al., Nucleic Acids Res., 13:3101-3109, 1985.

Jain, Cancer Meta. Rev., 9(3):253-266, 1990.

Jakeman et al., J. Clin. Invest., 89:244-253, 1992.

Johnson & Reithmuller, Hybridoma, 1:381, 1982.

Johnson et al., Am. J. Reprod. Immunol., 1:246, 1981

June et al., Molecular Cell Biology, 12:4472-4481, 1987.

June et al., Immunology Today, 11(6):211-216, 1990.

Jutila et al., J Exp Med, 175(6):1565-73, 1992.

Juweid et al., Cancer Res., 52:5144-5153, 1992.

Kabawat et al., Int. J. Gynecol. Pathol., 4:245, 1985.

Kabawat et al., Int. J. Gynecol. Pathol., 4:265, 1983.

Kandel et al., Cell, 66:1095-1104, 1991.

Kantor et al., Hybridoma, 1:473, 1982.

Karasek, J. Invest. Derm., 93(2):335-385, 1989.

Keelan et al., Am J Physiol, 266(1 Pt 2) pH278-90, January 1994a.

Keelan et al., J Nucl Med, 35(2):276-81, February 1994b.

Kennel et al., Cancer Res., 51:1529-1536, 1991.

Kim et. al., Growth Factors, 7:53-64, 1992.

Kim et al., Nature, 362:841-844, 1993.

Kimura et al., Immunogenetics, 11:373-381, 1983.

Kinsel et al., Cancer Res., 49:1052, 1989.

Kishimoto et al., Blood, 78(3):805-11, 1991.

Kisiel, J. Biol. Chem., 254(23):12230-12234, 1979.

Kjeldsen et al., 2nd Int. Wkshop of MAbs & Breast Cancer, San Fran.,November, 1986.

Klagsbrun & Folkman, Angiogenesis Handook of Experimental Pharmacology,Vol. 95, Sporn & Roberts, Springer-Verlag, Berlin, pp. 549-586, 1990.

Kohler & Milstein, Nature, 256:495-497, 1975.

Kohler & Milstein, Eur. J. Immunol., 6:511-519, 1976.

Kondo et al., Biochem. and Biophys. Res. Comm., 194(3):1234-1241, 1993.

Krishnaswamy et al., J. Biol. Chem., 267(36):26110-26120, 1992.

Krishnaswamy et al., J. Biol. Chem., 267(33):23696-23706, 1992.

Kufe et al., Hybridoma, 3:223, 1984.

Lan et al., Cancer Res., 44:1954, 1984.

Lan et al., Cancer Res., 45:305, 1985.

Lee et al., Methods in Enzymology, 237:146-164, 1994.

Leith et al., British J. Cancer, 66(2):345-8, 1992.

Lemkin et al., Proc. Am. Soc. Clin. Oncol., 3:47, 1984.

Leung et al., Science, 246:1306-1309, 1989.

Leydem et al., Cancer, 57:1135, 1986.

LoBuglio et al., JNCI, 80:932, 1988.

Logan et al., Proc. Natl. Acad. Sci. USA, 81:3655-3659, 1984.

Loop et al., Int. J. Cancer, 27:775, 1981.

Lord et al., In: Genetically Engineered Toxins, Frank (Ed.), M. DekkerPubl., p. 183, 1992.

Lowder et al., Blood, 69:199-210, 1987.

Lowe et al., Immunol Lett., 12:263-269, 1986.

Lowy et al., Cell, 22:817, 1980.

Maeda et al., J. Invest. Derm., 97:183-189, 1991.

Manabe et al., J. Lab. Clin. Med., 104(3):445-454, 1984.

Mandeville et al., Cancer Detect. Prev., 10:89, 1987.

Mann, TIBS 12, 229-233, 1987.

Mason & Williams, Biochem J, 187:1-20, 1980.

Massoglia et al., J. Cell. Phys., 132:531-537, 1987.

Masuko et al., Cancer Res., 44:2813, 1984.

Mattes et al., PNAS, 81:568, 1984.

Mazzocchi et al., Cancer Immunol. Immunother., 32:13-21, 1990.

McDicken et al., Br. J. Cancer, 52:59, 1985.

Menard et al., Cancer Res., 63:1295, 1983.

Messing et al., Third Cleveland Symposium on MacromoleculesandRecombinant DNA, Editor A. Walton, Elsevier, Amsterdam, 1981.

Metzelaar et al., Blood, 79(2):372-379, 1992.

Metzelaar et al., J. Biol. Chem., 266(5):3239-3245, 1991.

Mignatti et al., J. Cell. Biol., 113:1193-1201, 1991.

Millauer et al., Cell, 72:835-846, 1993.

Miotti et al., Cancer Res., 65:826, 1985.

Miotti et al., Int. J. Cancer, 39:297, 1987.

Montefort et al., Eur Respir J, 5(7):815-23, 1992.

Moroi and Aoki, J. Biol. Chem., 251(19):5956-5965, 1976.

Morrissey et al., Blood, 81:734-744, 1993.

Morrissey et al., Cell, 50:129-135, 1987.

Morrissey et al., Thrombosis Res., 52:247-261, 1988.

Moughal et al., J Periodontal Res, 27(6):623-30, 1992.

Mulligan et al., Proc. Natl. Acad. Sci. USA, 78:2072, 1981.

Mulligan et al., J. Clin. Invest., 88:1396-1406, 1991

Munz et al., J. Nucl, Med., 27:1739, 1986.

Murray et al., Radio. Onc., 16:221-234, 1989.

Nabel et al., Nature, 362:844-846, 1993.

Nakamura, Prog. Growth Factor Res., 3:67-86, 1991.

Nelson, 1991.

Nemerson, Blood, 71(1):1-8, 1988.

Neumann et al., Arch Dermatol, 130(7):879-83, 1994.

Nieuwenhuis et al., Blood, 70(3):838-845, 1987.

Nishikawa et al., Advances in Experimental Medicine and Biology,324:131-139, 1992.

Nitta et al., Lancet, 335:368-371, 1990.

Nolan & Kennedy, Biochemica et Biophysica Acta, 1040:1-11, 1990.

Norton et al., Biochem Biophys Res Commun, 195(1):250-8, 1993.

O'Connell et al., Clin. Exp. Immunol., 90:154-159, 1992.

O'Connell et al., J. Immunol., 144(2):521-525, 1990.

O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527, 1981.

Ogawa et al., British J. Haematology, 75:517-524, 1990.

Ohuchida et al. J. Am. Chem. Soc., 103(15):4597-4599, 1981.

Oi & Morrison, Mt. Sinai J. Med., 53(3):175-180, 1986.

Olander et al., Biochem. and Biophys. Res. Comm., 175(1):68-76, 1991.

Olofsson et al., Blood, 84(8):2749-58, 1994.

Osborn et al., Cell, 59:1203-1211, 1989.

Osterud et al., Thrombosis Res., 42:323-329, 1986.

Paborsky et al., J. Biol. Chem., 266(32):21911-21916, 1991.

Palleroni et al., Int. J. Cancer, 49:296-302, 1991.

Patt et al., Cancer Bull., 40:218, 1988.

Paul et al., Hybridoma, 5:171, 1986.

Paulus, Behrini Inst. Mitt., 78:118-132, 1985.

Perez et al., J. Exp. Med., 163:166-178, 1986.

Perez et al., J. Immunol., 137:2069-2072, 1986.

Perez et al., Nature, 316:354-356, 1985.

Perkins et al., Eur. J. Nucl. Med., 10:296, 1985.

Pietersz et al., Antibody, Immunoconj. Radiopharm., 1:79-103, 1988.

Pimm et al., J. Cancer Res. Clin. Oncol., 118:367-370, 1992.

Plate et al., Cancer Res., 53:5822-5827, 1993.

Plate et al., Nature, 359:845-848, 1992.

Pober et al., J. Exp. Med., 157:1339-1353, 1991.

Poels et al., J. Natl. Cancer Res., 44:4528, 1984.

Poels et al., J. Natl. Cancer, 76:781, 1986.

Pukrittayakamee et al., Mol. Biol. Med., 1:123-135, 1983.

Qian et al., Cancer Res., 140:3250, 1991.

Rao and Rapaport, Biochemistry, 85:6687-6691, 1988.

Rasmussen et al., Breast Cancer Res. Treat., 2:401, 1982.

Rehemtulla et al., Thrombosis and Haemostasis, 65(5):521-527, 1991.

Reilly et al., Biochem. Biophys. Res. Commun., 164:736-743, 1989.

Reisfeld et al., Melanoma Antigens and Antibodies, p. 317, 1982.

Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company,1980.

Rettig et al., Proc. Natl. Acad. Sci. USA, 89:10832-10836, 1992.

Riva et al., Int. J. Cancer, 2:114, 1988

Rivoltini et al., 3rd Int. Conf. Bispecific Antibodies and TargetedCellular Cytotoxicity, 1992.

Rowinksy, Clinical Investigation, Abstracts from Chemotherapy foundationsymposium X. Innovative cancer chemotherapy for tomorrow, pp. 6-9, 1992.

Ruco et al., Am. J Pathol., 137(5):1163-1171, 1990.

Ruf and Edgington, Thrombosis and Haemostasis, 66(5):529-533, 1991.

Ruf et al., J. Biol. Chem., 266(24):15719-15725, 1991.

Ruf et al., J. Biol. Chem., 266(4):2158-2166, 1991.

Ruf et al., JBC, 266:2158-2166, 1991.

Ruf & Edgington, FASEB J., 8:385-390, 1994.

Ruther et al., EMBO J., 2:1791, 1983.

Safran et al., Oncogene, 5:635-643, 1990.

Sainsbury et al., Lancet, 1:364, 1985.

Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y., 1989.

Sands, Immunoconjugates and Radiopharmaceuticals, 1:213-226, 1988.

Santerre et al., Gene, 30:147, 1984.

Saxton et al., Hybridoma, 1:433, 1982.

Scarpati et al., Biochemistry, 26:5234-5238, 1987.

Schlingemann et al., Lab. Invest., 52:71-76, 1985.

Schlingemann et al., Lab. Invest., 62:690-696, 1990.

Schlom et al., Adv. Cancer Res., 43:143, 1985.

Schutt et al., Immunol. Lett., 19:321-328, 1988.

Schweigerer et al., Nature, 325:257-259, 1987.

Sedmak et al., Transplantation, 58(12):1379-85, 1994.

Segal et al., 1992.

Senger et al., Cancer and Metastasis Reviews, 12:303-324, 1993.

Senger et al., Cancer Research, 50:1774-1778, 1990.

Shankar et al., J. Biol. Chem., 269(19):13936-13941, 1994.

Shen and Tai, J. Biol. Chem., 261(25):11585-11591, 1986.

Shepard et al., J. Clin. Immunol., 11:117-127, 1991.

Shockley et al., Ann. N.Y. Acad. Sci., 617:367-382, 1991.

Shrestha et. al., Eur. J. Cancer B. Oral. Oncol., 30B(6):393-9, 1994.

Shweiki et al., Nature, 359:843-847, 1992.

Silber et al., J Clin Invest, 93(4):1554-63, 1994.

Silverstein and Febbraio, Blood, 80(6):1470-1475, 1992.

Sioussat et al., Arch. Biochem. Biophys., 301(1):15-20, 1993.

Sloane, Cancer, 17:1786, 1981.

Smith et al., J. Virol., 46:584, 1983.

Smith et al., 1989.

Smith et al., Proc. Am. Soc. Clin. O. col., 6:250, 1987.

Soule et al., PNAS, 80:1332, 1983

Span et al., Immunology, 72(3):355-60, 1991.

Spicer et al., Proc. Natl. Acad. Sci. USA, 84:5148-5152, 1987.

Sporn et al., Blood, 81(9):2406-12, 1993.

Staerz et al., Nature, 314(6012):628-631, 1985.

Stavrou, Neurosurg. Rev., 13:7, 1990.

Stefanik et al., Cancer Research, 51:5760-5765, 1991.

Steinberg et al., J Heart Lung Transplant, 13(2):306-18, March-April1994.

Stern et al., Proc. Natl. Acad. Sci. USA, 80:4119-4123, 1982.

Stern et al., J. Biol. Chem., 260(11):6717-6722, 1985.

Stevenson et al., Chem. Immunol., 48:126-166, 1990.

Street et al., Cell. Immunol., 120:75-81, 1989.

Stuhlmiller et al., Hybridoma, 1:447, 1982.

Sugama et al., J. Cell Biol., 119(4):935-944, 1992.

Sunderland et al., Cancer Res., 44:4496, 1984.

Szybalska et al., Proc. Natl. Acad. Sci. USA, 48:2026, 1962.

Szymendera, Tumour Biology, 7:333, 1986.

Takahashi et al., Cancer, 61:881, 1988.

Teramoto et al., Cancer, 50:241, 1982.

Tessler et al., J. Biol. Chem., 269(17):12456-12461, 1994.

Thieme et al., Diabetes, 44(1):98-103, 1995.

Thompson et al., J. Natl. Cancer Inst., 70:409, 1983.

Thor et al., Cancer Res., 46:3118, 1986

Thorpe et al., Cancer Res., 48:6396-6403, 1988.

Ting et al., J. Immunol., 141:741-748, 1988.

Tischer et al., Biochem. and Biophys. Res. Comm., 165(3):1198-1206,1989.

Tischer et al., J. Biol. Chem., 266(18):11947-11954, 1991.

Titus et al., J. Immunol., 138:4018-4022, 1987.

Tomiyama et al., Blood, 79(9):2303-2312, 1992.

Tone et al., J. Biochem., 102(5):1033-1941, 1987.

Tsuji et al., Cancer Res., 45:2358, 1985.

Tuominen and Kallioinen, J. Cutan. Pathol. 21(5):424-9, 1994.

Tutt et al., Eur. J. Immunol., 21:1351-1358, 1991.

Ugarova et al., J. Biol. Chem., 268(28):21080-21087, 1993.

Ulich et al., Inflammation, 18(4):389-98, 1994.

Vaickus et al., Cancer Invest., 9:195-209, 1991.

Vaisman et al., J. Biol. Chem., 265(32):19461-19466, 1990.

Van Heeke et al., J. Biol. Chem., 264:5503-5509, 1989.

Van Duk et al., Int. J. Cancer, 43:344-349, 1989.

Veale et al., Arthritis Rheum, 36(7):893-900, 1993.

Venkateswaran et al., Hybridoma, 11(6):729-739, 1992.

Vitetta et al., Cancer Res., 15:4052-4058, 1991.

von Asmuth et al., Eur J Immunol, 22(10):2519-26, 1992.

Wagener et al., Int. J. Cancer, 33:469, 1984.

Wang et al., Int. J. Cancer, 54:363-370, 1993.

Wang et al., Biochem. and Biophys. Res. Comm., 177(1):286-291, 1991.

Warr et al., Blood, 75:1481-1489, 1990.

Watanabe et al., Proc. Natl. Acad. Sci. USA, 86:9456-9460, 1989.

Wawrzynczak & Thorpe, "Methods for preparing immunotoxins: effect of thelinkage on activity and stability", in: Immunoconiucates,: Antibodyconjugates in radioimaging and therapy of cancer, Vogel (ed), New York,Oxfod University Press, pp. 28-55, 1987.

Weiner et al., Cancer Res., 49:4062-4067, 1989.

Weiss et al., Blood, 73:968-975, 1989.

Whittle et al., Nature, 292:472-474, 1981.

Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567, 1980.

Wigler et al., Cell, 11:223, 1977.

Wildgoose et al., Blood, 80:25-28, 1992.

Williams and Esnouf, Biochem. J., 84:52-62, 1962.

Wilson et al., Int. J. Cancer, 28:293, 1981.

Wiman and Collen, Eur. J. Biochem., 78:19-26, 1977.

Wiman, Biochem. J., 191:229-232, 1980.

Winter & Milstein, Nature, 349:293-299, 1991.

Woodbury et al., PNAS, 77:2183, 1980.

Wu et al., Int. J. Pharm., 12:235-239, 1990.

Xu et al., J. Biol. Chem., 267(25):17792-17803, 1992.

Yamaguchi et al., Proc. Natl. Acad. Sci. USA, 91:484-488, 1994.

Yamaue et al., Biotherapy, 2:247-259, 1990.

Zamarron et al., J. Biol. Chem., 266(24):16193-16199, 1991.

Zhang et al., Int J Cancer, 59(6):823-9, 1994.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 27                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #             27   AGGC ACTACAA                                               - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #          32      TGAA TTCCCCCTTT CT                                         - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #                47GGTG CCTCGTGCTT CTGGCACTAC AAATACT                         - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #     38           CTCA GGCACTACAA ATACTGTG                                   - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #              50CTGGTG CCTCGTGGTT CTTGCGGCAC TACAAATACT                      - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #       35         CTCT GAATTCCCCC TTTCT                                      - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #              50CTGGTG CCTCGTGGTT GCGGAGGCGG TGGATCAGGC                      - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #       36         GAGG ATCCGCCACC TCCACT                                     - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #45                GCGG TGGAGGTAGT GGAGGTGGCG GATCC                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #          32      TGAA TTCCCCCTTT CT                                         - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #              50CTGGTG CCTCGTGGTT CTTGCGGCAC TACAAATACT                      - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 53 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - CGCGGATCCA CCGCCACCAG ATCCACCGCC TCCTTCTCTG AATTCCCCTT TC - #T                53                                                                          - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                # 44               GAGG CTCTTCAGGC ACTACAAATA CTGT                            - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #          31      TGAA TTCCCCTTTC T                                          - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #              50CTGGTG CCTCGTGGTT CTTGCGGCAC TACAAATACT                      - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #          31      TGAA TTCCCCTTTC T                                          - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                # 44               GGTG CCTCGTGGTT GCACTACAAA TACT                            - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #        34        CTCT GAATTCCCCT TTCT                                       - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                # 19               AAC                                                        - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "DNA"  (A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #       36         AATC TTCAGCTTCA GGAAAG                                     - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 657 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - TCAGGCACTA CAAATACTGT GGCAGCATAT AATTTAACTT GGAAATCAAC TA - #ATTTCAAG         60                                                                          - ACAATTTTGG AGTGGGAACC CAAACCCGTC AATCAAGTCT ACACTGTTCA AA - #TAAGCACT        120                                                                          - AAGTCAGGAG ATTGGAAAAG CAAATGCTTT TACACAACAG ACACAGAGTG TG - #ACCTCACC        180                                                                          - GACGAGATTG TGAAGGATGT GAAGCAGACG TACTTGGCAC GGGTCTTCTC CT - #ACCCGGCA        240                                                                          - GGGAATGTGG AGAGCACCGG TTCTGCTGGG GAGCCTCTGT ATGAGAACTC CC - #CAGAGTTC        300                                                                          - ACACCTTACC TGGAGACAAA CCTCGGACAG CCAACAATTC AGAGTTTTGA AC - #AGGTGGGA        360                                                                          - ACAAAAGTGA ATGTGACCGT AGAAGATGAA CGGACTTTAG TCAGAAGGAA CA - #ACACTTTC        420                                                                          - CTAAGCCTCC GGGATGTTTT TGGCAAGGAC TTAATTTATA CACTTTATTA TT - #GGAAATCT        480                                                                          - TCAAGTTCAG GAAAGAAAAC AGCCAAAACA AACACTAATG AGTTTTTGAT TG - #ATGTGGAT        540                                                                          - AAAGGAGAAA ACTACTGTTT CAGTGTTCAA GCAGTGATTC CCTCCCGAAC AG - #TTAACCGG        600                                                                          - AAGAGTACAG ACAGCCCGGT AGAGTGTATG GGCCAGGAGA AAGGGGAATT CA - #GAGAA           657                                                                          - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 219 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr As - #n Leu Thr Trp Lys Ser         #                15                                                           - Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pr - #o Lys Pro Val Asn Gln         #            30                                                               - Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gl - #y Asp Trp Lys Ser Lys         #        45                                                                   - Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Le - #u Thr Asp Glu Ile Val         #    60                                                                       - Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Va - #l Phe Ser Tyr Pro Ala         #80                                                                           - Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Gl - #u Pro Leu Tyr Glu Asn         #                95                                                           - Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr As - #n Leu Gly Gln Pro Thr         #           110                                                               - Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Va - #l Asn Val Thr Val Glu         #       125                                                                   - Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Th - #r Phe Leu Ser Leu Arg         #   140                                                                       - Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Le - #u Tyr Tyr Trp Lys Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr As - #n Thr Asn Glu Phe Leu         #               175                                                           - Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Ph - #e Ser Val Gln Ala Val         #           190                                                               - Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Th - #r Asp Ser Pro Val Glu         #       205                                                                   - Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gl - #u                             #   215                                                                       - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1947 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - TGCAGCTGCC TGGCTGCCTG GCCCTGGCTG CCCTGTGTAG CCTTGTGCAC AG - #CCAGCATG         60                                                                          - TGTTCCTGGC TCCTCAGCAA GCACGGTCGC TGCTCCAGCG GGTCCGGCGA GC - #CAACACCT        120                                                                          - TCTTGGAGGA GGTGCGCAAG GGCAACCTAG AGCGAGAGTG CGTGGAGGAG AC - #GTGCAGCT        180                                                                          - ACGAGGAGGC CTTCGAGGCT CTGGAGTCCT CCACGGCTAC GGATGTGTTC TG - #GGCCAAGT        240                                                                          - ACACAGCTTG TGAGACAGCG AGGACGCCTC GAGATAAGCT TGCTGCATGT CT - #GGAAGGTA        300                                                                          - ACTGTGCTGA GGGTCTGGGT ACGAACTACC GAGGGCATGT GAACATCACC CG - #GTCAGGCA        360                                                                          - TTGAGTGCCA GCTATGGAGG AGTCGCTACC CACATAAGCC TGAAATCAAC TC - #CACTACCC        420                                                                          - ATCCTGGGGC CGACCTACAG GAGAATTTCT GCCGCAACCC CGACAGCAGC AA - #CACGGGAC        480                                                                          - CCTGGTGCTA CACTACAGAC CCCACCGTGA GGAGGCAGGA ATGCAGCATC CC - #TGTCTGTG        540                                                                          - GCCAGGATCA AGTCACTGTA GCGATGACTC CACGCTCCGA AGGCTCCAGT GT - #GAATCTGT        600                                                                          - CACCTCCATT GGAGCAGTGT GTCCCTGATC GGGGGCAGCA GTACCAGGGG CG - #CCTGGCGG        660                                                                          - TGACCACACA TGGGCTCCCC TGCCTGGCCT GGGCCAGCGC ACAGGCCAAG GC - #CCTGAGCA        720                                                                          - AGCACCAGGA CTTCAACTCA GCTGTGCAGC TGGTGGAGAA CTTCTGCCGC AA - #CCCAGACG        780                                                                          - GGGATGAGGA GGGCGTGTGG TGCTATGTGG CCGGGAAGCC TGGCGACTTT GG - #GTACTGCG        840                                                                          - ACCTCAACTA TTGTGAGGAG GCCGTGGAGG AGGAGACAGG AGATGGGCTG GA - #TGAGGACT        900                                                                          - CAGACAGGGC CATCGAAGGG CGTACCGCCA CAAGTGAGTA CCAGACTTTC TT - #CAATCCGA        960                                                                          - GGACCTTTGG CTCGGGAGAG GCAGACTGTG GGCTGCGACC TCTGTTCGAG AA - #GAAGTCGC       1020                                                                          - TGGAGGACAA AACCGAAAGA GAGCTCCTGG AATCCTACAT CGACGGGCGC AT - #TGTGGAGG       1080                                                                          - GCTCGGATGC AGAGATCGGC ATGTCACCTT GGCAGGTGAT GCTTTTCCGG AA - #GAGTCCCC       1140                                                                          - AGGAGCTGCT GTGTGGGGCC AGCCTCATCA GTGACCGCTG GGTCCTCACC GC - #CGCCCACT       1200                                                                          - GCCTCCTGTA CCCGCCCTGG GACAAGAACT TCACCGAGAA TGACCTTCTG GT - #GCGCATTG       1260                                                                          - GCAAGCACTC CCGCACCAGG TACGAGCGAA ACATTGAAAA GATATCCATG TT - #GGAAAAGA       1320                                                                          - TCTACATCCA CCCCAGGTAC AACTGGCGGG AGAACCTGGA CCGGGACATT GC - #CCTGATGA       1380                                                                          - AGCTGAAGAA GCCTGTTGCC TTCAGTGACT ACATTCACCC TGTGTGTCTG CC - #CGACAGGG       1440                                                                          - AGACGGCAGC CAGCTTGCTC CAGGCTGGAT ACAAGGGGCG GGTGACAGGC TG - #GGGCAACC       1500                                                                          - TGAAGGAGAC GTGGACAGCC AACGTTGGTA AGGGGCAGCC CAGTGTCCTG CA - #GGTGGTGA       1560                                                                          - ACCTGCCCAT TGTGGAGCGG CCGGTCTGCA AGGACTCCAC CCGGATCCGC AT - #CACTGACA       1620                                                                          - ACATGTTCTG TGCTGGTTAC AAGCCTGATG AAGGGAAACG AGGGGATGCC TG - #TGAAGGTG       1680                                                                          - ACAGTGGGGG ACCCTTTGTC ATGAAGAGCC CCTTTAACAA CCGCTGGTAT CA - #AATGGGCA       1740                                                                          - TCGTCTCATG GGGTGAAGGC TGTGACCGGG ATGGGAAATA TGGCTTCTAC AC - #ACATGTGT       1800                                                                          - TCCGCCTGAA GAAGTGGATA CAGAAGGTCA TTGATCAGTT TGGAGAGTAG GG - #GGCCACTC       1860                                                                          - ATATTCTGGG CTCCTGGAAC CAATCCCGTG AAAGAATTAT TTTTGTGTTT CT - #AAAACTAT       1920                                                                          #           1947   GACT CTCAGCG                                               - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2462 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - TCAACAGGCA GGGGCAGCAC TGCAGAGATT TCATCATGGT CTCCCAGGCC CT - #CAGGCTCC         60                                                                          - TCTGCCTTCT GCTTGGGCTT CAGGGCTGCC TGGCTGCAGG CGGGGTCGCT AA - #GGCCTCAG        120                                                                          - GAGGAGAAAC ACGGGACATG CCGTGGAAGC CGGGGCCTCA CAGAGTCTTC GT - #AACCCAGG        180                                                                          - AGGAAGCCCA CGGCGTCCTG CACCGGCGCC GGCGCGCCAA CGCGTTCCTG GA - #GGAGCTGC        240                                                                          - GGCCGGGCTC CCTGGAGAGG GAGTGCAAGG AGGAGCAGTG CTCCTTCGAG GA - #GGCCCGGG        300                                                                          - AGATCTTCAA GGACGCGGAG AGGACGAAGC TGTTCTGGAT TTCTTACAGT GA - #TGGGGACC        360                                                                          - AGTGTGCCTC AAGTCCATGC CAGAATGGGG GCTCCTGCAA GGACCAGCTC CA - #GTCCTATA        420                                                                          - TCTGCTTCTG CCTCCCTGCC TTCGAGGGCC GGAACTGTGA GACGCACAAG GA - #TGACCAGC        480                                                                          - TGATCTGTGT GAACGAGAAC GGCGGCTGTG AGCAGTACTG CAGTGACCAC AC - #GGGCACCA        540                                                                          - AGCGCTCCTG TCGGTGCCAC GAGGGGTACT CTCTGCTGGC AGACGGGGTG TC - #CTGCACAC        600                                                                          - CCACAGTTGA ATATCCATGT GGAAAAATAC CTATTCTAGA AAAAAGAAAT GC - #CAGCAAAC        660                                                                          - CCCAAGGCCG AATTGTGGGG GGCAAGGTGT GCCCCAAAGG GGAGTGTCCA TG - #GCAGGTCC        720                                                                          - TGTTGTTGGT GAATGGAGCT CAGTTGTGTG GGGGGACCCT GATCAACACC AT - #CTGGGTGG        780                                                                          - TCTCCGCGGC CCACTGTTTC GACAAAATCA AGAACTGGAG GAACCTGATC GC - #GGTGCTGG        840                                                                          - GCGAGCACGA CCTCAGCGAG CACGACGGGG ATGAGCAGAG CCGGCGGGTG GC - #GCAGGTCA        900                                                                          - TCATCCCCAG CACGTACGTC CCGGGCACCA CCAACCACGA CATCGCGCTG CT - #CCGCCTGC        960                                                                          - ACCAGCCCGT GGTCCTCACT GACCATGTGG TGCCCCTCTG CCTGCCCGAA CG - #GACGTTCT       1020                                                                          - CTGAGAGGAC GCTGGCCTTC GTGCGCTTCT CATTGGTCAG CGGCTGGGGC CA - #GCTGCTGG       1080                                                                          - ACCGTGGCGC CACGGCCCTG GAGCTCATGG TGCTCAACGT GCCCCGGCTG AT - #GACCCAGG       1140                                                                          - ACTGCCTGCA GCAGTCACGG AAGGTGGGAG ACTCCCCAAA TATCACGGAG TA - #CATGTTCT       1200                                                                          - GTGCCGGCTA CTCGGATGGC AGCAAGGACT CCTGCAAGGG GGACAGTGGA GG - #CCCACATG       1260                                                                          - CCACCCACTA CCGGGGCACG TGGTACCTGA CGGGCATCGT CAGCTGGGGC CA - #GGGCTGCG       1320                                                                          - CAACCGTGGG CCACTTTGGG GTGTACACCA GGGTCTCCCA GTACATCGAG TG - #GCTGCAAA       1380                                                                          - AGCTCATGCG CTCAGAGCCA CGCCCAGGAG TCCTCCTGCG AGCCCCATTT CC - #CTAGCCCA       1440                                                                          - GCAGCCCTGG CCTGTGGAGA GAAAGCCAAG GCTGCGTCGA ACTGTCCTGG CA - #CCAAATCC       1500                                                                          - CATATATTCT TCTGCAGTTA ATGGGGTAGA GGAGGGCATG GGAGGGAGGG AG - #AGGTGGGG       1560                                                                          - AGGGAGACAG AGACAGAAAC AGAGAGAGAC AGAGACAGAG AGAGACTGAG GG - #AGAGACTC       1620                                                                          - TGAGGACATG GAGAGAGACT CAAAGAGACT CCAAGATTCA AAGAGACTAA TA - #GAGACACA       1680                                                                          - GAGATGGAAT AGAAAAGATG AGAGGCAGAG GCAGACAGGC GCTGGACAGA GG - #GGCAGGGG       1740                                                                          - AGTGCCAAGG TTGTCCTGGA GGCAGACAGC CCAGCTGAGC CTCCTTACCT CC - #CTTCAGCC       1800                                                                          - AAGCCCCACC TGCACGTGAT CTGCTGGCCC TCAGGCTGCT GCTCTGCCTT CA - #TTGCTGGA       1860                                                                          - GACAGTAGAG GCATGAACAC ACATGGATGC ACACACACAC ACGCCAATGC AC - #ACACACAG       1920                                                                          - AGATATGCAC ACACACGGAT GCACACACAG ATGGTCACAC AGAGATACGC AA - #ACACACCG       1980                                                                          - ATGCACACGC ACATAGAGAT ATGCACACAC AGATGCACAC ACAGATATAC AC - #ATGGATGC       2040                                                                          - ACGCACATGC CAATGCACGC ACACATCAGT GCACACGGAT GCACAGAGAT AT - #GCACACAC       2100                                                                          - CGATGTGCGC ACACACAGAT ATGCACACAC ATGGATGAGC ACACACACAC CA - #AGTGCGCA       2160                                                                          - CACACACCGA TGTACACACA CAGATGCACA CACAGATGCA CACACACCGA TG - #CTGACTCC       2220                                                                          - ATGTGTGCTG TCCTCTGAAG GCGGTTGTTT AGCTCTCACT TTTCTGGTTC TT - #ATCCATTA       2280                                                                          - TCATCTTCAC TTCAGACAAT TCAGAAGCAT CACCATGCAT GGTGGCGAAT GC - #CCCCAAAC       2340                                                                          - TCTCCCCCAA ATGTATTTCT CCCTTCGCTG GGTGCCGGGC TGCACAGACT AT - #TCCCCACC       2400                                                                          - TGCTTCCCAG CTTCACAATA AACGGCTGCG TCTCCTCCGC ACACCTGTGG TG - #CCTGCCAC       2460                                                                          #            2462                                                             - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1437 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - ATGCAGCGCG TGAACATGAT CATGGCAGAA TCACCAAGCC TCATCACCAT CT - #GCCTTTTA         60                                                                          - GGATATCTAC TCAGTGCTGA ATGTACAGTT TTTCTTGATC ATGAAAACGC CA - #ACAAAATT        120                                                                          - CTGAATCGGC CAAAGAGGTA TAATTCAGGT AAATTGGAAG AGTTTGTTCA AG - #GGAACCTT        180                                                                          - GAGAGAGAAT GTATGGAAGA AAAGTGTAGT TTTGAAGAAC CACGAGAAGT TT - #TTGAAAAC        240                                                                          - ACTGAAAAGA CAACTGAATT TTGGAAGCAG TATGTTGATG GAGATCAGTG TG - #AGTCCAAT        300                                                                          - CCATGTTTAA ATGGCGGCAG TTGCAAGGAT GACATTAATT CCTATGAATG TT - #GGTGTCCC        360                                                                          - TTTGGATTTG AAGGAAAGAA CTGTGAATTA GATGTAACAT GTAACATTAA GA - #ATGGCAGA        420                                                                          - TGCGAGCAGT TTTGTAAAAA TAGTGCTGAT AACAAGGTGG TTTGCTCCTG TA - #CTGAGGGA        480                                                                          - TATCGACTTG CAGAAAACCA GAAGTCCTGT GAACCAGCAG TGCCATTTCC AT - #GTGGAAGA        540                                                                          - GTTTCTGTTT CACAAACTTC TAAGCTCACC CGTGCTGAGG CTGTTTTTCC TG - #ATGTGGAC        600                                                                          - TATGTAAATC CTACTGAAGC TGAAACCATT TTGGATAACA TCACTCAAGG CA - #CCCAATCA        660                                                                          - TTTAATGACT TCACTCGGGT TGTTGGTGGA GAAGATGCCA AACCAGGTCA AT - #TCCCTTGG        720                                                                          - CAGGTTGTTT TGAATGGTAA AGTTGATGCA TTCTGTGGAG GCTCTATCGT TA - #ATGAAAAA        780                                                                          - TGGATTGTAA CTGCTGCCCA CTGTGTTGAA ACTGGTGTTA AAATTACAGT TG - #TCGCAGGT        840                                                                          - GAACATAATA TTGAGGAGAC AGAACATACA GAGCAAAAGC GAAATGTGAT TC - #GAGCAATT        900                                                                          - ATTCCTCACC ACAACTACAA TGCAGCTATT AATAAGTACA ACCATGACAT TG - #CCCTTCTG        960                                                                          - GAACTGGACG AACCCTTAGT GCTAAACAGC TACGTTACAC CTATTTGCAT TG - #CTGACAAG       1020                                                                          - GAATACACGA ACATCTTCCT CAAATTTGGA TCTGGCTATG TAAGTGGCTG GG - #CAAGAGTC       1080                                                                          - TTCCACAAAG GGAGATCAGC TTTAGTTCTT CAGTACCTTA GAGTTCCACT TG - #TTGACCGA       1140                                                                          - GCCACATGTC TTCGATCTAC AAAGTTCACC ATCTATAACA ACATGTTCTG TG - #CTGGCTTC       1200                                                                          - CATGAAGGAG GTAGAGATTC ATGTCAAGGA GATAGTGGGG GACCCCATGT TA - #CTGAAGTG       1260                                                                          - GAAGGGACCA GTTTCTTAAC TGGAATTATT AGCTGGGGTG AAGAGTGTGC AA - #TGAAAGGC       1320                                                                          - AAATATGGAA TATATACCAA GGTATCCCGG TATGTCAACT GGATTAAGGA AA - #AAACAAAG       1380                                                                          - CTCACTTAAT GAAAGATGGA TTTCCAAGGT TAATTCATTG GAATTGAAAA TT - #AACAG          1437                                                                          - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1126 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - GGATTCGAAG GCAAAAACTG TGAATTATTC ACACGGAAGC TCTGCAGCCT GG - #ACAACGGG         60                                                                          - GACTGTGACC AGTTCTGCCA CGAGGAACAG AACTCTGTGG TGTGCTCCTG CG - #CCCGCGGG        120                                                                          - TACACCCTGG CTGACAACGG CAAGGCCTGC ATTCCCACAG GGCCCTACCC CT - #GTGGGAAA        180                                                                          - CAGACCCTGG AACGCAGGAA GAGGTCAGTG GCCCAGGCCA CCAGCAGCAG CG - #GGGAGGCC        240                                                                          - CCTGACAGCA TCACATGGAA GCCATATGAT GCAGCCGACC TGGACCCCAC CG - #AGAACCCC        300                                                                          - TTCGACCTGC TTGACTTCAA CCAGACGCAG CCTGAGAGGG GCGACAACAA CC - #TCACCAGG        360                                                                          - ATCGTGGGAG GCCAGGAATG CAAGGACGGG GAGTGTCCCT GGCAGGCCCT GC - #TCATCAAT        420                                                                          - GAGGAAAACG AGGGTTTCTG TGGTGGAACC ATTCTGAGCG AGTTCTACAT CC - #TAACGGCA        480                                                                          - GCCCACTGTC TCTACCAAGC CAAGAGATTC GAAGGGGACC GGAACACGGA GC - #AGGAGGAG        540                                                                          - GGCGGTGAGG CGGTGCACGA GGTGGAGGTG GTCATCAAGC ACAACCGGTT CA - #CAAAGGAG        600                                                                          - ACCTATGACT TCGACATCGC CGTGCTCCGG CTCAAGACCC CCATCACCTT CC - #GCATGAAC        660                                                                          - GTGGCGCCTG CCTGCCTCCC CGAGCGTGAC TGGGCCGAGT CCACGCTGAT GA - #CGCAGAAG        720                                                                          - ACGGGGATTG TGAGCGGCTT CGGGCGCACC CACGAGAAGG GCCGGCAGTC CA - #CCAGGCTC        780                                                                          - AAGATGCTGG AGGTGCCCTA CGTGGACCGC AACAGCTGCA AGCTGTCCAG CA - #GCTTCATC        840                                                                          - ATCACCCAGA ACATGTTCTG TGCCGGCTAC GACACCAAGC AGGAGGATGC CT - #GCCAGGGG        900                                                                          - GACAGCGGGG GCCCGCACGT CACCCGCTTC AAGGACACCT ACTTCGTGAC AG - #GCATCGTC        960                                                                          - AGCTGGGGAG AGGGCTGTGC CCGTAAGGGG AAGTACGGGA TCTACACCAA GG - #TCACCGCC       1020                                                                          - TTCCTCAAGT GGATCGACAG GTCCATGAAA ACCAGGGGCT TGCCCAAGGC CA - #AGAGCCAT       1080                                                                          #               1126GTC CTCTCCATTA AAGTGAGATC CCACTC                          __________________________________________________________________________

What is claimed is:
 1. A method for delivering a coagulant totumor-associated vasculature, comprising administering to an animal witha vascularized tumor, a pharmaceutical composition comprising an amountof a binding ligand effective to promote blood coagulation in thetumor-associated vasculature, the binding ligand comprising:(a) a firstbinding region that binds to a surface-expressed, surface-accessible orsurface-localized component of a tumor cell, intratumoral vasculature ortumor-associated stroma, the first binding region operatively linked to(b) a coagulation factor or a second binding region that binds to acoagulation factor; wherein said second binding region comprises anantibody or an antigen binding region of an antibody.
 2. The method ofclaim 1, wherein said first binding region comprises an antibody or anantigen binding region of an antibody.
 3. The method of claim 2, whereinsaid first binding region comprises a monoclonal antibody or an antigenbinding region of a monoclonal antibody.
 4. The method of claim 2,wherein said first binding region comprises an antibody or an antigenbinding region of an antibody that binds to a surface-expressed,surface-accessible or surface-localized component of intratumoralvasculature.
 5. The method of claim 1, wherein said binding ligandcomprises a first binding region operatively linked to a coagulationfactor.
 6. The method of claim 1, wherein said binding ligand comprisesa first binding region operatively linked to a second binding regionthat binds to a coagulation factor.
 7. The method of claim 1, whereinsaid second binding region comprises an antigen binding region of anantibody that binds to a coagulation factor.
 8. The method of claim 1,wherein said first binding region is an antigen binding region of anantibody and said second binding region is an antigen binding region ofan antibody.
 9. The method of claim 1, wherein an exogenous coagulationfactor is delivered to said tumor-associated vasculature byadministering to said animal a binding ligand that comprises a firstbinding region covalently linked to a coagulation factor.
 10. The methodof claim 1, wherein an exogenous coagulation factor is delivered to saidtumor-associated vasculature by administering to said animal a bindingligand that comprises a first binding region covalently linked to asecond binding region that is non-covalently bound to a coagulationfactor.
 11. The method of claim 1, wherein an endogenous coagulationfactor is delivered to said tumor-associated vasculature byadministering to said animal a binding ligand that comprises a firstbinding region covalently linked to a second binding region that bindsto an endogenous coagulation factor, said binding ligand therebyconcentrating said endogenous coagulation factor at saidtumor-associated vasculature.
 12. The method of claim 1, wherein saidtumor-associated vasculature is associated with a benign vascularizedtumor.
 13. The method of claim 1, wherein said tumor-associatedvasculature is associated with a malignant vascularized tumor.
 14. Themethod of claim 1, wherein said binding ligand comprises the vitaminK-dependent coagulation Factor II/IIa, Factor VIINVIIa, Factor IX/IXa orFactor X/Xa.
 15. The method of claim 1, wherein said binding ligandcomprises a vitamin K-dependent coagulation factor lacking the Glamodification.
 16. The method of claim 1, wherein said binding ligandcomprises Tissue Factor or a Tissue Factor derivative.
 17. The method ofclaim 16, wherein said binding ligand comprises a mutant Tissue Factordeficient in the ability to activate Factor VII.
 18. The method of claim16, wherein said binding ligand comprises a truncated Tissue Factor. 19.The method of claim 16, wherein said binding ligand comprises a dimerictruncated Tissue Factor.
 20. The method of claim 1, wherein said bindingligand comprises Russell's viper venom Factor X activator, thromboxaneA₂, thromboxane A₂ synthase or α2-antiplasmin.
 21. A method for treatingcancer, comprising administering a pharmaceutical composition to ananimal with a vascularized tumor in an amount effective to promote bloodcoagulation in the vasculature of said tumor, wherein the pharmaceuticalcomposition comprises a coagulative binding ligand that comprises:(a) afirst binding region that binds to a surface-expressed,surface-accessible or surface-localized component of a tumor cell,intratumoral vasculature or tumor-associated stroma, the first bindingregion operatively linked to (b) a coagulation factor or a secondbinding region that binds to a coagulation factor; wherein said secondbinding region comprises an antibody or an antigen binding region of anantibody.
 22. The method of claim 21, wherein the first binding regionof said coagulative binding ligand comprises an antibody or an antigenbinding region of an antibody.
 23. The method of claim 22, wherein thefirst binding region of said coagulative binding ligand comprises amonoclonal antibody or an antigen binding region of a monoclonalantibody.
 24. The method of claim 22, wherein the first binding regionof said coagulative binding ligand comprises an IgG antibody or an IgMantibody.
 25. The method of claim 22, wherein the first binding regionof said coagulative binding ligand comprises an scFv, Fv, Fab', Fab orF(ab')₂ fragment of an antibody.
 26. The method of claim 21, wherein thesecond binding region of said coagulative binding ligand comprises anscFv, Fv, Fab', Fab or F(ab')₂ fragment of an antibody.
 27. The methodof claim 22, wherein the first binding region of said coagulativebinding ligand comprises an antibody or an antigen binding region of anantibody that binds to a cell surface antigen of a tumor cell.
 28. Themethod of claim 27, wherein the first binding region of said coagulativebinding ligand comprises an antibody or an antigen binding region of anantibody that binds to the tumor cell surface antigen p₁₈₅ ^(HER2), milkmucin core protein, TAG-72, Lewis a, carcinoembryonic antigen (CEA) or atumor cell surface antigen that binds to an antibody selected from thegroup consisting of B3 (ATCC HB 10573), KS1/4 (NRRL B-18356), 260F9(ATCC HB 8488), and D612 (ATCC HB 9796).
 29. The method of claim 22,wherein the first binding region of said coagulative binding ligandcomprises an antibody or an antigen binding region of an antibody thatbinds to a surface-expressed, surface-accessible or surface-localizedcomponent of intratumoral vasculature.
 30. The method of claim 29,wherein the first binding region of said coagulative binding ligandcomprises an antibody or an antigen binding region of an antibody thatbinds to a component of intratumoral vasculature that has little or noexpression at the surface of normal endothelial cells.
 31. The method ofclaim 22, wherein the first binding region of said coagulative bindingligand comprises an antibody or an antigen binding region of an antibodythat binds to an intratumoral vasculature endothelial cell surfacemolecule.
 32. The method of claim 31, wherein the first binding regionof said coagulative binding ligand comprises an antibody or an antigenbinding region of an antibody that binds to an MHC Class II protein, aVEGF/VPF receptor, an FGF receptor, a TGFβ receptor, a TIE, VCAM-1,ICAM-1, P-selectin, E-selectin, α_(v) β₃ integrin, pleiotropin,endosialin or endoglin.
 33. The method of claim 32, wherein the firstbinding region of said coagulative binding ligand comprises an antibodyor an antigen binding region of an antibody that binds to an MHC ClassII protein.
 34. The method of claim 32, wherein the first binding regionof said coagulative binding ligand comprises an antibody or an antigenbinding region of an antibody that binds to a VEGF/VPF receptor.
 35. Themethod of claim 32, wherein the first binding region of said coagulativebinding ligand comprises an antibody or an antigen binding region of anantibody that binds to VCAM-1.
 36. The method of claim 32, wherein thefirst binding region of said coagulative binding ligand comprises anantibody or an antigen binding region of an antibody that binds toICAM-1.
 37. The method of claim 32, wherein the first binding region ofsaid coagulative binding ligand comprises an antibody or an antigenbinding region of an antibody that binds to P-selectin.
 38. The methodof claim 32, wherein the first binding region of said coagulativebinding ligand comprises an antibody or an antigen binding region of anantibody that binds to E-selectin.
 39. The method of claim 32, whereinthe first binding region of said coagulative binding ligand comprises anantibody or an antigen binding region of an antibody that binds toendoglin.
 40. The method of claim 39, wherein the first binding regionof said coagulative binding ligand comprises an antibody or an antigenbinding region of an antibody that binds to the same epitope as themonoclonal antibody TEC-4 (ATCC HB 12312) or to the same epitope as themonoclonal antibody TEC-11 (ATCC HB 12311).
 41. The method of claim 29,wherein the first binding region of said coagulative binding ligandcomprises an antibody or an antigen binding region of an antibody thatbinds to a ligand or growth factor that is bound to an intratumoralvasculature cell surface molecule.
 42. The method of claim 41, whereinthe first binding region of said coagulative binding ligand comprises anantibody or an antigen binding region of an antibody that binds to FGF,TGFβ, a ligand that binds to a TIE, a tumor-associated fibronectinisoform, scatter factor/hepatocyte growth factor (HGF), platelet factor4 (PF4), PDGF or TIMP.
 43. The method of claim 41, wherein the firstbinding region of said coagulative binding ligand comprises an antibodyor an antigen binding region of an antibody that binds to the sameepitope as the monoclonal antibody GV39 (ATCC HB 12450) or to the sameepitope as the monoclonal antibody GV97 (ATCC HB 12451).
 44. The methodof claim 41, wherein the first binding region of said coagulativebinding ligand comprises an antibody or an antigen binding region of anantibody that binds to FGF.
 45. The method of claim 41, wherein thefirst binding region of said coagulative binding ligand comprises anantibody or an antigen binding region of an antibody that binds to TGFβ.46. The method of claim 29, wherein the first binding region of saidcoagulative binding ligand comprises an antibody or an antigen bindingregion of an antibody that binds to a ligand:receptor complex or agrowth factor:receptor complex, but does not bind to the ligand orgrowth factor or to the receptor when the ligand or growth factor or thereceptor is not in the ligand:receptor complex.
 47. The method of claim22, wherein the first binding region of said coagulative binding ligandcomprises an antibody or an antigen binding region of an antibody thatbinds to a component of tumor stroma.
 48. The method of claim 47,wherein the first binding region of said coagulative binding ligandcomprises an antibody or an antigen binding region of an antibody thatbinds to a basement membrane component, tenascin, an activated plateletor an antigen inducible on a basement membrane component or activatedplatelet.
 49. The method of claim 21, wherein the first binding regionof said coagulative binding ligand comprises a ligand or receptor thatbinds to a surface-expressed, surface-accessible or surface-localizedcomponent of a tumor cell, intratumoral vasculature or tumor-associatedstroma.
 50. The method of claim 49, wherein the first binding region ofsaid coagulative binding ligand comprises VEGF/VPF, FGF, TGFβ, a ligandthat binds to a TIE, a tumor-associated fibronectin isoform, scatterfactor, hepatocyte growth factor (HGF), platelet factor 4 (PF4), PDGF,TIMP or a soluble binding domain of a VEGF/VPF receptor.
 51. The methodof claim 21, wherein said coagulative binding ligand comprises a firstbinding region that is operatively linked to a coagulation factor. 52.The method of claim 21, wherein said coagulative binding ligandcomprises a first binding region that is operatively linked to a secondbinding region that binds to a coagulation factor.
 53. The method ofclaim 51, wherein said coagulative binding ligand is a bispecificantibody.
 54. The method of claim 21, wherein said coagulative bindingligand comprises the vitamin K-dependent coagulation Factor II/IIa,Factor VII/VIIa, Factor IX/IXa or Factor X/Xa.
 55. The method of claim21, wherein said coagulative binding ligand comprises a vitaminK-dependent coagulation factor lacking the Gla modification.
 56. Themethod of claim 21, wherein said coagulative binding ligand comprisesTissue Factor or a Tissue Factor derivative.
 57. The method of claim 56,wherein said coagulative binding ligand comprises a mutant Tissue Factordeficient in the ability to activate Factor VII.
 58. The method of claim57, wherein said coagulative binding ligand comprises a mutant TissueFactor that includes a mutation in the amino acid region between aboutposition 157 and about position
 167. 59. The method of claim 58, whereinsaid coagulative binding ligand comprises a mutant Tissue Factor inwhich Trp at position 158 is changed to Arg; wherein Ser at position 162is changed to Ala; wherein Gly at position 164 is changed to Ala; orwherein Trp at position 158 is changed to Arg and Ser at position 162 ischanged to Ala.
 60. The method of claim 56, wherein said coagulativebinding ligand comprises a truncated Tissue Factor.
 61. The method ofclaim 60, wherein said coagulative binding ligand comprises a truncatedTissue Factor that consists essentially of amino acids 1-219 of themature Tissue Factor protein.
 62. The method of claim 60, wherein saidcoagulative binding ligand comprises a truncated Tissue Factor that hasthe amino acid sequence of SEQ ID NO:23.
 63. The method of claim 56,wherein said coagulative binding ligand comprises a dimeric TissueFactor.
 64. The method of claim 63, wherein said coagulative bindingligand comprises a polymeric Tissue Factor.
 65. The method of claim 56,wherein said coagulative binding ligand comprises a Tissue Factorderivative with a hydrophobic membrane insertion moiety, said TissueFactor derivative being linked to said first binding region via abiologically-releasable bond.
 66. The method of claim 21, wherein saidcoagulative binding ligand comprises Russell's viper venom Factor Xactivator, thromboxane A₂, thromboxane A₂ synthase or α2-antiplasmin.67. The method of claim 22, wherein the first binding region of saidcoagulative binding ligand comprises a humanized antibody or an antigenbinding region thereof.
 68. The method of claim 21, wherein saidcoagulation factor is a human coagulation factor.
 69. The method ofclaim 68, wherein said coagulation factor is a human Tissue Factor orTissue Factor derivative.
 70. The method of claim 21, wherein the firstbinding region of said coagulative binding ligand is operatively linkedto said coagulation factor or said second binding region via across-linker.
 71. The method of claim 21, wherein said coagulativebinding ligand is a fusion protein prepared by expressing a recombinantvector in a host cell, wherein the vector comprises, in the same readingframe, a DNA segment encoding said first binding region operativelylinked to a DNA segment encoding said coagulation factor or said secondbinding region.
 72. The method of claim 21, wherein the second bindingregion is bound to an exogenous coagulation factor prior toadministration to said animal and wherein said coagulative bindingligand delivers said exogenous coagulation factor to the vasculature ofsaid tumor.
 73. The method of claim 21, wherein the second bindingregion is not bound to a coagulation factor prior to administration tosaid animal and wherein said coagulative binding ligand binds to anendogenous coagulation factor and the binding ligand thereby deliversthe endogenous coagulation factor to the vasculature of said tumor. 74.The method of claim 21, wherein said pharmaceutical composition isadministered to said animal parenterally.
 75. The method of claim 21,wherein said pharmaceutical composition is administered to said animalby injection into the vascularized tumor site.
 76. The method of claim21, wherein said animal is a human patient.
 77. A method for treatingcancer, comprising administering a pharmaceutical composition to ananimal with a vascularized tumor in an amount effective to promotespecific blood coagulation in the vasculature of said tumor relative tothe vasculature in non-tumor sites, wherein the pharmaceuticalcomposition comprises a coagulative binding ligand that comprises:(a) afirst antibody or antigen binding region of an antibody that binds to asurface-expressed, surface-accessible or surface-localized component ofintratumoral vasculature or tumor-associated stroma; the first antibodyor antigen binding region of an antibody operatively linked to (b) acoagulation factor or a second antibody or antigen binding region of anantibody that binds to a coagulation factor.
 78. The method of claim 77,wherein said coagulative binding ligand comprises a first antibody orantigen binding region of an antibody operatively linked to acoagulation factor.
 79. The method of claim 77, wherein said coagulativebinding ligand comprises a first antibody or antigen binding region ofan antibody operatively linked to a second antibody or antigen bindingregion of an antibody that binds to a coagulation factor.
 80. The methodof claim 77, wherein said coagulative binding ligand comprises a firstantibody or antigen binding region of an antibody that binds to asurface-expressed, surface-accessible or surface-localized component ofthe intratumoral vasculature of a vascularized tumor.
 81. The method ofclaim 77, wherein said coagulative binding ligand comprises TissueFactor or a Tissue Factor derivative.
 82. The method of claim 77,wherein said first binding region comprises a humanized antibody or anantigen binding region thereof.
 83. The method of claim 77, wherein saidcoagulation factor is a human coagulation factor.
 84. A method fortreating cancer, comprising administering a pharmaceutical compositionto an animal with a vascularized tumor in an amount effective to promoteblood coagulation in the vasculature of said tumor, wherein thepharmaceutical composition comprises a coagulative binding ligand thatcomprises:(a) a first binding region that binds to a surface-expressed,surface-accessible or surface-localized component of intratumoral bloodvessels of a vascularized tumor; the first binding region operativelylinked to (b) a coagulant or an antibody or an antigen binding region ofan antibody that binds to a coagulant.
 85. A method for treating cancer,comprising administering a pharmaceutical composition to an animal witha vascularized tumor in an amount effective to promote specific bloodcoagulation in the vasculature of said tumor relative to the vasculaturein non-tumor sites, wherein the pharmaceutical composition comprises acoagulative binding ligand that comprises:(a) a first antibody orantigen binding region of an antibody that binds to a surface-expressed,surface-accessible or surface-localized component of intratumoral bloodvessels of a vascularized tumor; the first antibody or antigen bindingregion thereof operatively linked to (b) a coagulant or a secondantibody or antigen binding region of an antibody that binds to acoagulant.
 86. A method for treating cancer, comprising administering apharmaceutical composition to an animal with a vascularized tumor in anamount effective to promote specific blood coagulation in thevasculature of said tumor relative to the vasculature in non-tumorsites, wherein the pharmaceutical composition comprises a coagulativebinding ligand that comprises:(a) a first antibody or antigen bindingregion of an antibody that binds to a surface-expressed,surface-accessible or surface-localized component of intratumoral bloodvessels of a vascularized tumor; the first antibody or antigen bindingregion thereof operatively linked to (b) a human Tissue Factor or humanTissue Factor derivative or to a second antibody or antigen bindingregion of an antibody that binds to a human Tissue Factor or derivativethereof.
 87. A method for treating cancer, comprising administering apharmaceutical composition to an animal with a vascularized tumor in anamount effective to promote specific blood coagulation in thevasculature of said tumor relative to the vasculature in non-tumorsites, wherein the pharmaceutical composition comprises a coagulativebinding ligand that comprises:(a) a first antibody or antigen bindingregion of an antibody that binds to a surface-expressed,surface-accessible or surface-localized component of intratumoral bloodvessels of a vascularized tumor; the first antibody or antigen bindingregion thereof operatively linked to (b) a second antibody or antigenbinding region of an antibody that binds to a human Tissue Factor orderivative thereof.